Combination therapy for the treatment of ocular neovascular disorders

ABSTRACT

The invention features methods for treating a patient diagnosed with, or at risk of developing, a neovascular disorder by administering a PDGF antagonist and a VEGF antagonist to the patient. The invention also features a pharmaceutical composition containing a PDGF antagonist and a VEGF antagonist for the treatment or prevention of a neovascular disorder.

RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application Ser.No. 60/556,837, filed Mar. 26, 2004, which is hereby incorporated in itsentirety by reference.

FIELD OF THE INVENTION

This invention relates to the fields of ophthalmology and medicine. Morespecifically, this invention relates to the treatment of neovasculardisorders of the eye using a combination of agents that inhibit bothplatelet-derived growth factor (PDGF) and vascular endothelial growthfactor (VEGF).

BACKGROUND OF THE INVENTION

Angiogenesis, also called neovascularization, involves the formation ofsprouts from preexistent blood vessels and their invasion intosurrounding tissue. A related process, vasculogenesis, involves thedifferentiation of endothelial cells and angioblasts that are alreadypresent throughout a tissue, and their subsequent linking together toform blood vessels.

Angiogenesis occurs extensively during development, and also occurs inthe healthy body during wound healing in order to restore blood flow totissues after injury or insult. Angiogenesis, however, has also beenimplicated in cancer and tumor formation. Indeed, the quantity of bloodvessels in a tumor tissue is a strong negative prognostic indicator inbreast cancer (Weidner et al., (1992) J. Natl. Cancer Inst.84:1875-1887), prostate cancer (Weidner et al., (1993) Am. J. Pathol.143:401-409), brain tumors (Li et al., (1994) Lancet 344:82-86), andmelanoma (Foss et al., (1996) Cancer Res. 56:2900-2903). Angiogenesishas also recently been implicated in other disease states in many areasof medicine, including rheumatology, dermatology, cardiology andophthalmology. In particular, undesirable or pathologicaltissue-specific angiogenesis has been associated with certain specificdisease states including rheumatoid arthritis, atherosclerosis, andpsoriasis (see e.g., Fan et al., (1995) Trends Pharmacol. Sci. 16: 57;and Folkman (1995) Nature Med. 1: 27). Furthermore, the alteration ofvascular permeability is thought to play a role in both normal andpathological physiological processes (Cullinan-Bove et al., (1993)Endocrinol. 133: 829; Senger et al., (1993) Cancer and MetastasisReviews 12: 303). Although the angiogenic process in each of thesediseases is likely to share many features with developmentalangiogenesis and tumor angiogenesis, each may also have unique aspectsconferred by the influence of surrounding cells.

Several ocular disorders involve alterations in angiogenesis. Forexample, diabetic retinopathy, the third leading cause of adultblindness (accounting for almost 7% of blindness in the USA), isassociated with extensive angiogenic events. Nonproliferativeretinopathy is accompanied by the selective loss of pericytes within theretina, and their loss results in dilation of associated capillariesdilation and a resulting increase in blood flow. In the dilatedcapillaries, endothelial cells proliferate and form outpouchings, whichbecome microaneurysms, and the adjacent capillaries become blocked sothat the area of retina surrounding these microaneurysms is notperfused. Eventually, shunt vessels appear between adjacent areas ofmicro aneurysms, and the clinical picture of early diabetic retinopathywith micro aneurysms and areas of nonperfused retina is seen. Themicroaneurysms leak and capillary vessels may bleed, causing exudatesand hemorrhages. Once the initial stages of background diabeticretinopathy are established, the condition progresses over a period ofyears, developing into proliferative diabetic retinopathy and blindnessin about 5% of cases. Proliferative diabetic retinopathy occurs whensome areas of the retina continue losing their capillary vessels andbecome nonperfused, leading to the appearance of new vessels on the diskand elsewhere on the retina. These new blood vessels grow into thevitreous and bleed easily, leading to preretinal hemorrhages. Inadvanced proliferative diabetic retinopathy, a massive vitreoushemorrhage may fill a major portion of the vitreous cavity. In addition,the new vessels are accompanied by fibrous tissue proliferation that canlead to traction retinal detachment.

Diabetic retinopathy is associated primarily with the duration ofdiabetes mellitus; therefore, as the population ages and diabeticpatients live longer, the prevalence of diabetic retinopathy willincrease. Laser therapy is currently used in both nonproliferative andproliferative diabetic retinopathy. Focal laser treatment of the leakingmicroaneurysms surrounding the macular area reduces visual loss in 50%of patients with clinically significant macular edema. In proliferativediabetic retinopathy, panretinal photocoagulation results in severalthousand tiny burns scattered throughout the retina (sparing the maculararea); this treatment reduces the rate of blindness by 60 percent. Earlytreatment of macular edema and proliferative diabetic retinopathyprevents blindness for 5 years in 95% of patients, whereas latetreatment prevents blindness in only 50 percent. Therefore, earlydiagnosis and treatment are essential.

Another ocular disorder involving neovascularization is age-relatedmacular degeneration (AMD), a disease that affects approximately one inten Americans over the age of 65. AMD is characterized by a series ofpathologic changes in the macula, the central region of the retina,which is accompanied by decreased visual acuity, particularly affectingcentral vision. AMD involves the single layer of cells called theretinal pigment epithelium that lies immediately beneath the sensoryretina. These cells nourish and support the portion of the retina incontact with them, i.e., the photoreceptor cells that contain the visualpigments. The retinal pigment epithelium lies on the Bruch membrane, abasement membrane complex which, in AMD, thickens and becomes sclerotic.New blood vessels may break through the Bruch membrane from theunderlying choroid, which contains a rich vascular bed. These vesselsmay in turn leak fluid or bleed beneath the retinal pigment epitheliumand also between the retinal pigment epithelium and the sensory retina.Subsequent fibrous scarring disrupts the nourishment of thephotoreceptor cells and leads to their death, resulting in a loss ofcentral visual acuity. This type of age-related maculopathy is calledthe “wet” type because of the leaking vessels and the subretinal edemaor blood. The wet type accounts for only 10% of age-related maculopathycases but results in 90% of cases of legal blindness from maculardegeneration in the elderly. The “dry” type of age-related maculopathyinvolves disintegration of the retinal pigment epithelium along withloss of the overlying photoreceptor cells. The dry type reduces visionbut usually only to levels of 20/50 to 20/100.

AMD is accompanied by distortion of central vision with objectsappearing larger or smaller or straight lines appearing distorted, bent,or without a central segment. In the wet type of AMD, a small detachmentof the sensory retina may be noted in the macular area, but thedefinitive diagnosis of a subretinal neovascular membrane requiresfluorescein angiography. In the dry type, drusen may disturb thepigmentation pattern in the macular area. Drusen are excrescences of thebasement membrane of the retinal pigment epithelium that protrude intothe cells, causing them to bulge anteriorly; their role as a risk factorin age-related maculopathy is unclear. No treatment currently exists forthe dry type of age-related maculopathy. Laser treatment is used in thewet type of age-related maculopathy and initially obliterates theneovascular membrane and prevents further visual loss in about 50% ofpatients at 18 months. By 60 months, however, only 20% still have asubstantial benefit.

Multiple molecular mediators of angiogenesis have been identifiedincluding basic and acidic fibroblast growth factors (aFGF, bFGF),transforming growth factors alpha and beta (TGFα, TGFβ),platelet-derived growth factor (PDGF), angiogenin, platelet-derivedendothelial cell growth factor (PD-ECGF), interleukin-8 (IL-8), andvascular endothelial growth factor (VEGF). Other stimulators implicatedin angiogenesis include angiopoietin-1, Del-1, follistatin, granulocytecolony-stimulating factor (G-CSF), hepatocyte growth factor (HGF),leptin, midkine, placental growth factor, pleiotrophin (PTN),progranulin, proliferin, and tumor necrosis factor-alpha (TNF-alpha). Inaddition, control of angiogenesis is further mediated by a number ofnegative regulators of angiogenesis produced by the body includingangioarrestin, ngiostatin (plasminogen fragment), antiangiogenicantithrombin III, cartilage-derived inhibitor (CDI), CD59 complementfragment, endostatin (collagen XVIII fragment), fibronectin fragment,gro-beta, heparinases, heparin hexasaccharide fragment, human chorionicgonadotropin (hCG), interferon alpha/beta/gamma, interferon inducibleprotein (IP-10), interleukin-12, kringle 5 (plasminogen fragment),metalloproteinase inhibitors (TIMPs), 2-methoxyestradiol, placentalribonuclease inhibitor, plasminogen activator inhibitor, plateletfactor-4 (PF4), prolactin 16 kD fragment, proliferin-related protein(PRP), retinoids, tetrahydrocortisol-S, thrombospondin-1 (TSP-1),vasculostatin, and vasostatin (calreticulin fragment).

Among these angiogenic regulators, VEGF appears to play a key role as apositive regulator of the abnormal angiogenesis accompanying tumorgrowth (reviewed in Brown et al., (1996) Control of Angiogenesis(Goldberg and Rosen, eds.) Birkhauser, Basel, and Thomas (1996) J. Biol.Chem. 271:603-606). Furthermore, recently the role of the PDGF-B memberof the PDGF family of signaling molecules has been under investigation,since it appears to play a role in the formation, expansion and properfunction of perivascular cells, sometimes referred to as mural cells,e.g., vascular smooth muscle, mesangial cells, and pericytes.

While much has been learned about angiogenesis, or neovascularization,accompanying development, wound healing and tumor formation, it remainsto be determined whether there are differences between these forms ofangiogenesis and ocular angiogenesis. Significantly, while angiogenesisaccompanying, e.g., collateral blood vessel formation in the heart, maybe beneficial and adaptive to the organism, pathological ocularneovascularization accompany, e.g., AMD, has no known benefit and oftenleads to blindness (for review, see Campochiaro (2000) J. Cell. Physiol.184: 301-10). Therefore, although advances in the understanding of themolecular events accompanying neovascularization have been made, thereexists a need to utilize this understanding to develop further methodsfor treating neovascular diseases disorders, including ocularneovascular diseases and disorders such as the choroidalneovascularization that occurs with AMD and diabetic retinopathy.

SUMMARY OF THE INVENTION

It has been discovered that the combination of anti-VEGF and anti-PDGFagents surprisingly affords synergistic therapeutic benefits fortreating an ocular neovascular disease.

Accordingly, the invention features a method for treating a patientdiagnosed with or at risk for developing a neovascular disorder. Thismethod includes administering to the patient an anti-VEGF agent and ananti-PDGF agent as a primary or adjunct therapeutic treatment.

In one aspect, the invention provides a method for suppressing aneovascular disorder in a patient in need thereof, by administering tothe patient a PDGF antagonist and a VEGF antagonist, simultaneously, orwithin about 90 days of each other, in amounts sufficient to suppressthe neovascular disorder in the patient.

In another aspect, the invention provides a method for treating apatient diagnosed with, or at risk for developing, a neovasculardisorder in a patient in need thereof, by administering to the patient aPDGF antagonist and a VEGF antagonist, simultaneously or within 90 daysof each other, in amounts sufficient to treat the patient.

In particular embodiments of these aspects, the method of the inventioninvolves administering the PDGF antagonist and the VEGF antagonistwithin about 10 days of each other. In another embodiment of the methodof the invention, the PDGF antagonist and the VEGF antagonist areadministered within 5 days of each other. In yet another embodiment ofthe method of the invention, the PDGF antagonist and the VEGF antagonistare administered within about 24 hours of each other. In a particularembodiment of the method of the invention, the PDGF antagonist and saidVEGF antagonist are administered simultaneously.

In another embodiment, the method of the invention involvesadministration of a PDGF antagonist that is a PDGF-B antagonist. Instill another embodiment, the method of the invention involvesadministration of a VEGF antagonist that is a VEGF-A antagonist.

In certain embodiments, the method of the invention involvesadministration of a PDGF antagonist that is a nucleic acid molecule, anaptamer, an antisense RNA molecule, a ribozyme, an RNAi molecule, aprotein, a peptide, a cyclic peptide, an antibody, a binding fragment ofan antibody fragment, a sugar, a polymer, or a small organic compound.In another embodiment, the method of the invention involvesadministration of a VEGF antagonist that is a nucleic acid molecule, anaptamer, an antisense RNA molecule, a ribozyme, an RNAi molecule, aprotein, a peptide, a cyclic peptide, an antibody, a binding fragment ofan antibody fragment, a sugar, a polymer, or a small organic compound.

In a particular embodiment, the method of the invention involvesadministration of a VEGF antagonist that is an aptamer, such as anEYE001 aptamer. In another embodiment, the method of the inventioninvolves administration of a VEGF antagonist that is an antibody orbinding fragment thereof.

In a particular embodiment, the method of the invention involvesadministration of a PDGF antagonist that is an aptamer, an antibody or abinding fragment thereof. In another particular embodiment, the methodof the invention involves administration of a PDGF antagonist that is anantisense oligonucleotide.

In yet another embodiment of this aspect of the invention, the PDGFantagonist and/or the VEGF antagonist are pro-drugs.

In one embodiment, the method of the invention provides a means forsuppressing or treating an ocular neovascular disorder. In someembodiments, ocular neovascular disorders amenable to treatment orsuppression by the method of the invention include ischemic retinopathy,iris neovascularization, intraocular neovascularization, age-relatedmacular degeneration, corneal neovascularization, retinalneovascularization, choroidal neovascularization, diabetic retinalischemia, or proliferative diabetic retinopathy. In still anotherembodiment, the method of the invention provides a means for suppressingor treating psoriasis or rheumatoid arthritis in a patient in needthereof or a patient diagnosed with or at risk for developing such adisorder.

The invention also provides a pharmaceutical composition that includesboth a PDGF antagonist and a VEGF antagonist, as well a pharmaceuticallyacceptable carrier. In this aspect, the PDGF and VEGF antagonists arepresent both in amounts sufficient to suppress the neovascular disorderin the patient.

In one embodiment of this aspect, the pharmaceutical compositionincludes a PDGF antagonist that is a PDGF-B antagonist. In anotherembodiment, the pharmaceutical composition includes a VEGF antagonistthat is a VEGF-A antagonist.

In certain embodiments, the pharmaceutical composition of the inventionincludes a PDGF antagonist that is a nucleic acid molecule, an aptamer,an antisense RNA molecule, a ribozyme, an RNAi molecule, a protein, apeptide, a cyclic peptide, an antibody, a binding fragment of anantibody fragment, a sugar, a polymer or a small organic compound. Inanother embodiment, the pharmaceutical composition of the inventionincludes a VEGF antagonist that is a nucleic acid molecule, an aptamer,an antisense RNA molecule, a ribozyme, an RNAi molecule, a protein, apeptide, a cyclic peptide, an antibody, a binding fragment of anantibody fragment, a sugar, a polymer, or a small organic compound.

In other particular embodiments, the pharmaceutical composition of theinvention includes a VEGF antagonist that is an aptamer, such as anEYE001 aptamer. In one embodiment, the pharmaceutical composition of theinvention includes a VEGF antagonist that is an antibody or bindingfragment thereof.

In a particular embodiment, the pharmaceutical composition of theinvention includes a PDGF antagonist that is an antibody or bindingfragment thereof. In another particular embodiment, the pharmaceuticalcomposition of the invention includes a PDGF antagonist that is anantisense oligonucleotide.

The pharmaceutical composition the invention may include apharmaceutically acceptable carrier which includes a microsphere or ahydrogel formulation.

In yet another embodiment, the PDGF antagonist and/or the VEGFantagonist are pro-drugs.

In another embodiment, the pharmaceutical composition of the inventionprovides a means for suppressing or treating an ocular neovasculardisorder. In some embodiments, ocular neovascular disorders amenable totreatment or suppression by the pharmaceutical composition of theinvention include ischemic retinopathy, iris neovascularization,intraocular neovascularization, age-related macular degeneration,corneal neovascularization, retinal neovascularization, choroidalneovascularization, diabetic retinal ischemia, or proliferative diabeticretinopathy. In still other embodiments, the pharmaceutical compositionof the invention provides a means for suppressing or treating psoriasisor rheumatoid arthritis in a patient in need thereof, or a patientdiagnosed with or at risk for developing such a disorder.

The invention also provides a pharmaceutical pack that includes both aPDGF antagonist and a VEGF antagonist. In one embodiment of this aspect,the pharmaceutical pack includes a PDGF antagonist that is a PDGF-Bantagonist. In another embodiment of this aspect, the pharmaceuticalpack includes a VEGF antagonist that is a VEGF-A antagonist.

In another embodiment, the PDGF antagonist and VEGF antagonist of thepharmaceutical pack are formulated separately and in individual dosageamounts. In still another embodiment, the PDGF antagonist and VEGFantagonist of the pharmaceutical pack are formulated together.

In some particular embodiments, the pharmaceutical pack of the inventionincludes a VEGF antagonist that is an aptamer, such as an EYE001aptamer. In other embodiments, the pharmaceutical pack of the inventionincludes a VEGF antagonist that is an antibody or binding fragmentthereof.

In some embodiments, the pharmaceutical pack of the invention includes aPDGF antagonist that is an antibody or binding fragment thereof. Inother particular embodiment, the pharmaceutical pack of the inventionincludes a PDGF antagonist that is an antisense oligonucleotide. In yetanother embodiment of this aspect, the PDGF antagonist and/or the VEGFantagonist are pro-drugs.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1(A) is a schematic representation of the nucleic acid sequence ofa human PDGF-B (GenBank Accession No. X02811) (SEQ ID NO: 1).

FIG. 1(B) is a schematic representation of the amino acid sequence of ahuman PDGF-B (GenBank Accession No. CAA26579) (SEQ ID NO: 2).

FIG. 1(C) is a schematic representation of the nucleic acid sequence ofa human PDGF-A (GenBank Accession No. X06374) (SEQ ID NO: 11).

FIG. 1(D) is a schematic representation of the polypeptide sequence of ahuman PDGF-A (GenBank Accession No. CAA29677) (SEQ ID NO: 12).

FIG. 2(A) is a schematic representation of the nucleic acid sequence ofa human VEGF (GenBank Accession No: NM_(—)003376) (SEQ ID NO: 3).

FIG. 2(B) is a schematic representation of the amino acid sequence of ahuman VEGF polypeptide (GenBank Accession No. NP_(—)003367) (SEQ ID NO:4).

FIG. 3(A) is a schematic representation of the nucleic acid sequence ofa human PDGFR-B (GenBank Accession No. NM_(—)002609) (SEQ ID NO: 5).

FIG. 3(B) is a schematic representation of the polypeptide sequence of ahuman PDGFR-B (GenBank Accession No. NP_(—)002600) (SEQ ID NO: 6).

FIG. 3(C) is a schematic representation of the nucleic acid sequence ofa human PDGFR-A (GenBank Accession No. NM_(—)006206) (SEQ ID NO: 13).

FIG. 3(D) is a schematic representation of the polypeptide sequence of ahuman PDGFR-A (GenBank Accession No. NP_(—)006197) (SEQ ID NO: 14).

FIG. 4(A) is a schematic representation of the nucleic acid sequence ofa human VEGFR-1 (Flt-1) (GenBank Accession No. AF063657) (SEQ ID NO: 7).

FIG. 4(B) is schematic a representation of the polypeptide sequence of ahuman VEGFR-1 (Flt-1) (GenBank Accession No.) (SEQ ID NO: 8).

FIG. 4(C) is a schematic representation of the nucleic acid sequence ofa human VEGFR-2 (KDR/Flk-1) (GenBank Accession No. AF035121) (SEQ ID NO:9).

FIG. 4(D) is a schematic representation of the polypeptide sequence of ahuman VEGFR-2 (KDR/Flk-1) (GenBank Accession No. AAB88005) (SEQ ID NO:10).

FIG. 5 is a graphical representation of the results of a cornealneovascularization assay comparing a control treatment (cont), Gleevectreatment (an anti-PDGF agent), and Macugen™ treatment (i.e. pegaptanibtreatment, an anti-VEGF agent), to the results of a combinationtreatment with Macugen™ and Gleevec (anti-PDGF/anti-VEGF combinationtherapy).

FIG. 6(A) is a photographic representation of a fluorescent-microscopicimage of corneal neovascularization occurring in control (PEG-treated)mouse cornea.

FIG. 6(B) is a photographic representation of a fluorescent-microscopicimage of corneal neovascularization occurring in a Gleevec-treated mousecornea.

FIG. 6(C) is a photographic representation of a fluorescent-microscopicimage of corneal neovascularization occurring in a Macugen™-treatedmouse cornea.

FIG. 6(D) is a photographic representation of a fluorescent-microscopicimage of corneal neovascularization occurring in a mouse cornea treatedwith both Macugen™ and Gleevec.

FIG. 7(A) is a photographic representation of a fluorescent-microscopicimage showing that normal corneal vasculature is unaffected byadministration of APB5 (PDGFR antibody, an anti-PDGF agent).

FIG. 7(B) is a photographic representation of a fluorescent-microscopicimage showing that normal corneal vasculature is unaffected byadministration of Gleevec.

FIG. 7(C) is a photographic representation of a fluorescent-microscopicimage showing that normal corneal vasculature is unaffected byadministration of Macugen™ (Mac) and Gleevec together.

FIG. 7(D) is a photographic representation of a fluorescent-microscopicimage showing that normal corneal vasculature is unaffected byadministration of PEG.

FIG. 8 is a graphical representation of the results of a laser-inducedchoroidal neovascularization assay comparing a control treatment (cont),Gleevec treatment (an anti-PDGF agent), and Macugen™ treatment (i.e.pegaptanib treatment, an anti-VEGF agent), to the results of acombination treatment with Macugen™ and Gleevec (anti-PDGF/anti-VEGFcombination therapy).

FIG. 9 is a graphical representation of the results of a laser-inducedchoroidal neovascularization assay comparing a control-treated (cont),APB5-treated (an anti-PGFR antibody, which acts as an anti-PDGF agent),and Macugen treatment (i.e. pegaptanib treatment, an anti-VEGF aptamer),to the results of a combination treatment with Macugen and APB5(Mac+APB5).

FIG. 10 is a graphical representation of the results of a retinaldevelopmental model comparing a control treatment (cont), ARC-127treatment (an anti-PDGF agent), and Macugen treatment (i.e. pegaptanibtreatment, an anti-VEGF agent), to the results of a combinationtreatment with Macugen and ARC-127 (anti-PDGF/anti-VEGF combinationtherapy).

FIG. 11 is a graphical representation of the results of a cornealneovascularization assay comparing a control treatment (cont), ARC-127treatment (an anti-PDGF agent), and Macugen treatment (i.e. pegaptanibtreatment, an anti-VEGF agent), to the results of a combinationtreatment with Macugen and ARC-127 (anti-PDGF/anti-VEGF combinationtherapy).

FIG. 12(A) is a photographic representation of a fluorescent-microscopicimage of corneal neovascularization occurring in control mouse cornea.

FIG. 12(B) is a photographic representation of a fluorescent-microscopicimage of corneal neovascularization occurring in a ARC-127-treated mousecornea.

FIG. 12(C) is a photographic representation of a fluorescent-microscopicimage of corneal neovascularization occurring in a Macugen-treated mousecornea.

FIG. 12(D) is a photographic representation of a fluorescent-microscopicimage of corneal neovascularization occurring in a mouse cornea treatedwith both Macugen and ARC-127.

FIG. 13 is a graphical representation of the results of a cornealneovascularization assay comparing a control treatment (cont), APB-5treatment (an anti-PDGF agent), and Macugen treatment (i.e. pegaptanibtreatment, an anti-VEGF agent), to the results of a combinationtreatment with Macugen and APB-5 (anti-PDGF/anti-VEGF combinationtherapy).

FIG. 14 is a graphical representation of the results of a cornealneovascularization assay comparing a control treatment (cont), APB-5treatment (an anti-PDGF agent), and Macugen treatment (i.e. pegaptanibtreatment, an anti-VEGF agent), to the results of a combinationtreatment with Macugen and APB-5 (anti-PDGF/anti-VEGF combinationtherapy).

DETAILED DESCRIPTION OF THE INVENTION

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference.

Definitions

As used herein, the following terms and phrases shall have the meaningsset forth below. Unless defined otherwise, all technical and scientificterms used herein have the same meaning as commonly understood to one ofordinary skill in the art to which this invention belongs.

By “antagonist” is meant an agent that inhibits, either partially orfully, the activity or production of a target molecule. In particular,the term “antagonist,” as applied selectively herein, means an agentcapable of decreasing levels of PDGF, PDGFR, VEGF or VEGFR geneexpression, mRNA levels, protein levels or protein activity. Exemplaryforms of antagonists include, for example, proteins, polypeptides,peptides (such as cyclic peptides), antibodies or antibody fragments,peptide mimetics, nucleic acid molecules, antisense molecules,ribozymes, aptamers, RNAi molecules, and small organic molecules.Exemplary non-limiting mechanisms of antagonist inhibition of theVEGF/VEGFR and PDGF/PDGFR ligand/receptor targets include repression ofligand synthesis and/or stability (e.g., using, antisense, ribozymes orRNAi compositions targeting the ligand gene/nucleic acid), blocking ofbinding of the ligand to its cognate receptor (e.g., using anti-ligandaptamers, antibodies or a soluble, decoy cognate receptor), repressionof receptor synthesis and/or stability (e.g., using, antisense,ribozymes or RNAi compositions targeting the ligand receptorgene/nucleic acid), blocking of the binding of the receptor to itscognate receptor (e.g., using receptor antibodies) and blocking of theactivation of the receptor by its cognate ligand (e.g., using receptortyrosine kinase inhibitors). In addition, the antagonist may directly orindirectly inhibit the target molecule.

The term “antibody” as used herein is intended to include wholeantibodies, e.g., of any isotype (IgG, IgA, IgM, IgE, etc.), andincludes fragments thereof which recognize and are also specificallyreactive with vertebrate (e.g., mammalian) protein, carbohydrates, etc.Antibodies can be fragmented using conventional techniques and thefragments screened for utility in the same manner as described above forwhole antibodies. Thus, the term includes segments of proteolyticallycleaved or recombinantly-prepared portions of an antibody molecule thatare capable of selectively reacting with a certain protein. Non-limitingexamples of such proteolytic and/or recombinant fragments include Fab,F(ab′)2, Fab′, Fv, and single chain antibodies (scFv) containing a V[L]and/or V[H] domain joined by a peptide linker. The scFv's may becovalently or noncovalently linked to form antibodies having two or morebinding sites. The subject invention includes polyclonal, monoclonal, orother purified preparations of antibodies and recombinant antibodies.

The term “aptamer,” used herein interchangeably with the term “nucleicacid ligand,” means a nucleic acid that, through its ability to adopt aspecific three dimensional conformation, binds to and has anantagonizing (i.e., inhibitory) effect on a target. The target of thepresent invention is PDGF or VEGF (or one of their cognate receptorsPDGFR or VEGFR), and hence the term PDGF aptamer or nucleic acid ligandor VEGF aptamer or nucleic acid ligand (or PDGFR aptamer or nucleic acidligand or VEGFR aptamer or nucleic acid ligand) is used. Inhibition ofthe target by the aptamer may occur by binding of the target, bycatalytically altering the target, by reacting with the target in a waywhich modifies/alters the target or the functional activity of thetarget, by covalently attaching to the target as in a suicide inhibitor,by facilitating the reaction between the target and another molecule.Aptamers may be comprised of multiple ribonucleotide units,deoxyribonucleotide units, or a mixture of both types of nucleotideresidues. Aptamers may further comprise one or more modified bases,sugars or phosphate backbone units as described in further detailherein.

By “antibody antagonist” is meant an antibody molecule as herein definedwhich is able to block or significantly reduce one or more activities ofa target PDGF or VEGF. For example, an VEGF inhibitory antibody mayinhibit or reduce the ability of VEGF to stimulate angiogenesis.

A nucleotide sequence is “complementary” to another nucleotide sequenceif each of the bases of the two sequences matches, i.e., are capable offorming Watson Crick base pairs. The term “complementary strand” is usedherein interchangeably with the term “complement.” The complement of anucleic acid strand can be the complement of a coding strand or thecomplement of a non-coding strand.

The phrases “conserved residue” “or conservative amino acidsubstitution” refer to grouping of amino acids on the basis of certaincommon properties. A functional way to define common properties betweenindividual amino acids is to analyze the normalized frequencies of aminoacid changes between corresponding proteins of homologous organisms.According to such analyses, groups of amino acids may be defined whereamino acids within a group exchange preferentially with each other, andtherefore resemble each other most in their impact on the overallprotein structure (Schulz, G. E. and R. H. Schirmer, Principles ofProtein Structure, Springer-Verlag). Examples of amino acid groupsdefined in this manner include:

-   (i) a charged group, consisting of Glu and Asp, Lys, Arg and His,-   (ii) a positively-charged group, consisting of Lys, Arg and His,-   (iii) a negatively-charged group, consisting of Glu and Asp,-   (iv) an aromatic group, consisting of Phe, Tyr and Trp,-   (v) a nitrogen ring group, consisting of His and Trp,-   (vi) a large aliphatic nonpolar group, consisting of Val, Leu and    Ile,-   (vii) a slightly-polar group, consisting of Met and Cys,-   (viii) a small-residue group, consisting of Ser, Thr, Asp, Asn, Gly,    Ala, Glu, Gln and Pro,-   (ix) an aliphatic group consisting of Val, Leu, Ile, Met and Cys,    and-   (x) a small hydroxyl group consisting of Ser and Thr.

In addition to the groups presented above, each amino acid residue mayform its own group, and the group formed by an individual amino acid maybe referred to simply by the one and/or three letter abbreviation forthat amino acid commonly used in the art.

The term “interact” as used herein is meant to include detectablerelationships or association (e.g., biochemical interactions) betweenmolecules, such as interaction between protein-protein, protein-nucleicacid, nucleic acid-nucleic acid, and protein-small molecule or nucleicacid-small molecule in nature.

The term “interacting protein” refers to protein capable of interacting,binding, and/or otherwise associating to a protein of interest, such asfor example a PDGF or a VEGF protein, or their corresponding cognatereceptors.

The term “isolated” as used herein with respect to nucleic acids, suchas DNA or RNA, refers to molecules separated from other DNAs, or RNAs,respectively that are present in the natural source of themacromolecule. Similarly the term “isolated” as used herein with respectto polypeptides refers to protein molecules separated from otherproteins that are present in the source of the polypeptide. The termisolated as used herein also refers to a nucleic acid or peptide that issubstantially free of cellular material, viral material, or culturemedium when produced by recombinant DNA techniques, or chemicalprecursors or other chemicals when chemically synthesized.

“Isolated nucleic acid” is meant to include nucleic acid fragments,which are not naturally occurring as fragments and would not be found inthe natural state. The term “isolated” is also used herein to refer topolypeptides, which are isolated from other cellular proteins and ismeant to encompass both purified and recombinant polypeptides.

As used herein, the terms “label” and “detectable label” refer to amolecule capable of detection, including, but not limited to,radioactive isotopes, fluorophores, chemiluminescent moieties, enzymes,enzyme substrates, enzyme cofactors, enzyme inhibitors, dyes, metalions, ligands (e.g., biotin or haptens) and the like. The term“fluorescer” refers to a substance or a portion thereof, which iscapable of exhibiting fluorescence in the detectable range. Particularexamples of labels which may be used under the invention includefluorescein, rhodamine, dansyl, umbelliferone, Texas red, luminol,NADPH, alpha-beta-galactosidase and horseradish peroxidase.

The “level of expression of a gene in a cell” refers to the level ofmRNA, as well as pre-mRNA nascent transcript(s), transcript processingintermediates, mature mRNA(s) and degradation products, encoded by thegene in the cell, as well as the level of protein translated from thatgene.

As used herein, the term “nucleic acid” refers to polynucleotides suchas deoxyribonucleic acid (DNA), and, where appropriate, ribonucleic acid(RNA). The term should also be understood to include, as equivalents,analogs of either RNA or DNA made from nucleotide analogs, and, asapplicable to the embodiment being described, single (sense orantisense) and double-stranded polynucleotides, ESTs, chromosomes,cDNAs, mRNAs, and rRNAs are representative examples of molecules thatmay be referred to as nucleic acids.

The term “oligonucleotide” refers to an oligomer or polymer ofnucleotide or nucleoside monomers consisting of naturally occurringbases, sugars and intersugar (backbone) linkages. The term also includesmodified or substituted oligomers comprising non-naturally occurringmonomers or portions thereof, which function similarly. Incorporation ofsubstituted oligomers is based on factors including enhanced cellularuptake, or increased nuclease resistance and are chosen as is known inthe art. The entire oligonucleotide or only portions thereof may containthe substituted oligomers.

The term “percent identical” refers to sequence identity between twoamino acid sequences or between two nucleotide sequences. Identity caneach be determined by comparing a position in each sequence, which maybe aligned for purposes of comparison. When an equivalent position inthe compared sequences is occupied by the same base or amino acid, thenthe molecules are identical at that position; when the equivalent siteoccupied by the same or a similar amino acid residue (e.g., similar insteric and/or electronic nature), then the molecules can be referred toas homologous (similar) at that position. Expression as a percentage ofhomology, similarity, or identity refers to a function of the number ofidentical or similar amino acids at positions shared by the comparedsequences. Various alignment algorithms and/or programs may be used,including Hidden Markov Model (HMM), FASTA and BLAST. HNiM, FASTA andBLAST are available through the National Center for BiotechnologyInformation, National Library of Medicine, National Institutes ofHealth, Bethesda, Md. and the European Bioinformatic Institute EBI. Inone embodiment, the percent identity of two sequences that can bedetermined by these GCG programs with a gap weight of 1, e.g., eachamino acid gap is weighted as if it were a single amino acid ornucleotide mismatch between the two sequences. Other techniques foralignment are described in Methods in Enzymology, vol. 266: ComputerMethods for Macromolecular Sequence Analysis (1996), ed. Doolittle,Academic Press, Inc., a division of Harcourt Brace & Co., San Diego,Calif., USA. Where desirable, an alignment program that permits gaps inthe sequence is utilized to align the sequences. The Smith Waterman isone type of algorithm that permits gaps in sequence alignments (see(1997) Meth. Mol. Biol. 70: 173-187). Also, the GAP program using theNeedleman and Wunsch alignment method can be utilized to alignsequences. More techniques and algorithms including use of the HMM aredescribed in Sequence Structure and Databanks: A Practical Approach(2000), ed. Oxford University Press, Incorporated and in Bioinformatics:Databases and Systems (1999) ed. Kluwer Academic Publishers. Analternative search strategy uses MPSRCH software, which runs on a MASPARcomputer. MPSRCH uses a Smith-Watermnan algorithm to score sequences ona massively parallel computer. This approach improves ability to pick updistantly related matches, and is especially tolerant of small gaps andnucleotide sequence errors. Nucleic acid-encoded amino acid sequencescan be used to search both protein and DNA databases. Databases withindividual sequences are described in Methods in Enzymology, ed.Doolittle, supra. Databases include Genbank, EMBL, and DNA Database ofJapan (DDBJ).

“Perfectly matched” in reference to a duplex means that the poly- oroligonucleotide strands making up the duplex form a double strandedstructure with one other such that every nucleotide in each strandundergoes Watson-Crick basepairing with a nucleotide in the otherstrand. The term also comprehends the pairing of nucleoside analogs,such as deoxyinosine, nucleosides with 2-aminopurine bases, and thelike, that may be employed. A mismatch in a duplex between a targetpolynucleotide and an oligonucleotide or polynucleotide means that apair of nucleotides in the duplex fails to undergo Watson-Crick bonding.In reference to a triplex, the term means that the triplex consists of aperfectly matched duplex and a third strand in which every nucleotideundergoes Hoogsteen or reverse Hoogsteen association with a base pair ofthe perfectly matched duplex.

The term “RNA interference,” “RNAi,” or “siRNA” all refer to any methodby which expression of a gene or gene product is decreased byintroducing into a target cell one or more double-stranded RNAs, whichare homologous to the gene of interest (particularly to the messengerRNA of the gene of interest, e.g., PDGF or VEGF).

Polymorphic variants also may encompass “single nucleotidepolymorphisms” (SNPs) in which the polynucleotide sequence varies by onebase (e.g., a one base variation in PDGF or VEGF). The presence of SNPsmay be indicative of, for example, a certain population, a diseasestate, or a propensity for a disease state.

The “profile” of an aberrant, e.g., tumor cell's biological state refersto the levels of various constituents of a cell that change in responseto the disease state. Constituents of a cell include levels of RNA,levels of protein abundances, or protein activity levels.

The term “protein” is used interchangeably herein with the terms“peptide” and “polypeptide.” The term “recombinant protein” refers to aprotein of the present invention which is produced by recombinant DNAtechniques, wherein generally DNA encoding the expressed protein or RNAis inserted into a suitable expression vector which is in turn used totransform a host cell to produce the heterologous protein or RNA.Moreover, the phrase “derived from,” with respect to a recombinant geneencoding the recombinant protein is meant to include within the meaningof “recombinant protein” those proteins having an amino acid sequence ofa native protein, or an amino acid sequence similar thereto which isgenerated by mutations, including substitutions and deletions, of anaturally occurring protein.

As used herein, the term “transgene” means a nucleic acid sequence(encoding, e.g., one of the target nucleic acids, or an antisensetranscript thereto), which has been introduced into a cell. A transgenecould be partly or entirely heterologous, i.e., foreign, to thetransgenic animal or cell into which it is introduced, or, is homologousto an endogenous gene of the transgenic animal or cell into which it isintroduced, but which is designed to be inserted, or is inserted, intothe animal's genome in such a way as to alter the genome of the cellinto which it is inserted (e.g., it is inserted at a location whichdiffers from that of the natural gene or its insertion results in aknockout). A transgene can also be present in a cell in the form of anepisome. A transgene can include one or more transcriptional regulatorysequences and any other nucleic acid, such as introns, that may benecessary for optimal expression of a selected nucleic acid.

By “neovascular disorder” is meant a disorder characterized by alteredor unregulated angiogenesis other than one accompanying oncogenic orneoplastic transformation, i.e., cancer. Examples of neovasculardisorders include psoriasis, rheumatoid arthritis, and ocularneovascular disorders including diabetic retinopathy and age-relatedmacular degeneration.

As used herein, the terms “neovascularization” and “angiogenesis” areused interchangeably. Neovascularization and angiogenesis refer to thegeneration of new blood vessels into cells, tissue, or organs. Thecontrol of angiogenesis is typically altered in certain disease statesand, in many cases, the pathological damage associated with the diseaseis related to altered, unregulated, or uncontrolled angiogenesis.Persistent, unregulated angiogenesis occurs in a multiplicity of diseasestates, including those characterized by the abnormal growth byendothelial cells, and supports the pathological damage seen in theseconditions including leakage and permeability of blood vessels.

By “ocular neovascular disorder” is meant a-disorder characterized byaltered or unregulated angiogenesis in the eye of a patient. Exemplaryocular neovascular disorders include optic disc neovascularization, irisneovascularization, retinal neovascularization, choroidalneovascularization, corneal neovascularization, vitrealneovascularization, glaucoma, pannus, pterygium, macular edema, diabeticretinopathy, diabetic macular edema, vascular retinopathy, retinaldegeneration, uveitis, inflammatory diseases of the retina, andproliferative vitreoretinopathy.

The term “treating” a neovascular disease in a subject or “treating” asubject having a neovascular disease refers to subjecting the subject toa pharmaceutical treatment, e.g., the administration of a drug, suchthat at least one symptom of the neovascular disease is decreased.Accordingly, the term “treating” as used herein is intended to encompasscuring as well as ameliorating at least one symptom of the neovascularcondition or disease. Accordingly, “treating” as used herein, includesadministering or prescribing a pharmaceutical composition for thetreatment or prevention of an ocular neovascular disorder.

By “patient” is meant any animal. The term “animal” includes mammals,including, but is not limited to, humans and other primates. The termalso includes domesticated animals, such as cows, hogs, sheep, horses,dogs, and cats.

By “PDGF” or “platelet-derived growth factor” is meant a mammalianplatelet-derived growth factor that affects angiogenesis or anangiogenic process. As used herein, the term “PDGF” includes the varioussubtypes of PDGF including PDGF-B (see FIGS. 1(A) and (B)), and PDGF-A(see FIGS. 1(C) and (D)). Further, as used herein, the term “PDGF”refers to PDGF-related angiogenic factors such as PDGF-C and PDGF-D thatact through a cognate PDGF receptor to stimulate angiogenesis or anangiogenic process. In particular, the term “PDGF” means any member ofthe class of growth factors that (i) bind to a PDGF receptor such asPDGFR-B (see FIGS. 3(A) and (B)), or PDGFR-A (see FIGS. 3(C) and (D));(ii) activates a tyrosine kinase activity associated with the VEGFreceptor; and (iii) thereby affects angiogenesis or an angiogenicprocess. As used herein, the term “PDGF” generally refers to thosemembers of the class of growth factors that induce DNA synthesis andmitogenesis through the binding and activation of a platelet-derivedgrowth factor cell surface receptor (i.e., PDGFR) on a responsive celltype. PDGFs effect specific biological effects including, for example:directed cell migration (chemotaxis) and cell activation; phospholipaseactivation; increased phosphatidylinositol turnover and prostaglandinmetabolism; stimulation of both collagen and collagenase synthesis byresponsive cells; alteration of cellular metabolic activities, includingmatrix synthesis, cytokine production, and lipoprotein uptake;induction, indirectly, of a proliferative response in cells lacking PDGFreceptors; and potent vasoconstrictor activity. The term “PDGF” is meantto include both a “PDGF” polypeptide and its corresponding “PDGF”encoding gene or nucleic acid.

By “PDGF-A” is meant an A chain polypeptide of PDGF and itscorresponding encoding gene or nucleic acid.

By “PDGF-B” is meant a B chain polypeptide of PDGF and its correspondingencoding gene or nucleic acid.

By “VEGF,” or “vascular endothelial growth factor,” is meant a mammalianvascular endothelial growth factor that affects angiogenesis or anangiogenic process. As used herein, the term “VEGF” includes the varioussubtypes of VEGF (also known as vascular permeability factor (VPF) andVEGF-A) (see FIGS. 2(A) and (B)) that arise by, e.g., alternativesplicing of the VEGF-A/VPF gene including VEGF₁₂₁, VEGF₁₆₅ and VEGF₁₈₉.Further, as used herein, the term “VEGF” refers to VEGF-relatedangiogenic factors such as PIGF (placenta growth factor), VEGF-B,VEGF-C, VEGF-D and VEGF-E that act through a cognate VEFG receptor tostimulate angiogenesis or an angiogenic process. In particular, the term“VEGF” means any member of the class of growth factors that (i) bind toa VEGF receptor such as VEGFR-1 (Flt-1) (see FIGS. 4(A) and (B)),VEGFR-2 (KDR/Flk-1) (see FIGS. 4(C) and (D)), or VEGFR-3 (FLT-4); (ii)activates a tyrosine kinase activity associated with the VEGF receptor;and (iii) thereby affects angiogenesis or an angiogenic process. Theterm “VEGF” is meant to include both a “VEGF” polypeptide and itscorresponding “VEGF” encoding gene or nucleic acid.

By “PDGF antagonist” is meant an agent that reduces, or inhibits, eitherpartially or fully, the activity or production of a PDGF. A PDGFantagonist may directly or indirectly reduce or inhibit a specific PDGFsuch as PDGF-B. Furthermore, “PDGF antagonists” consistent with theabove definition of “antagonist,” may include agents that act on eithera PDGF ligand or its cognate receptor so as to reduce or inhibit aPDGF-associated receptor signal. Examples of such “PDGF antagonists”thus include, for example: antisense, ribozymes or RNAi compositionstargeting a PDGF nucleic acid; anti-PDGF aptamers, anti-PDGF antibodiesor soluble PDGF receptor decoys that prevent binding of a PDGF to itscognate receptor; antisense, ribozymes or RNAi compositions targeting acognate PDGF receptor (PDGFR) nucleic acid; anti-PDGFR aptamers oranti-PDGFR antibodies that bind to a cognate PDGFR receptor; and PDGFRtyrosine kinase inhibitors.

By “VEGF antagonist” is meant an agent that reduces, or inhibits, eitherpartially or fully, the activity or production of a VEGF. A VEGFantagonist may directly or indirectly reduce or inhibit a specific VEGFsuch as VEGF₁₆₅. Furthermore, “VEGF antagonists” consistent with theabove definition of “antagonist,” may include agents that act on eithera VEGF ligand or its cognate receptor so as to reduce or inhibit aVEGF-associated receptor signal. Examples of such “VEGF antagonists”thus include, for example: antisense, ribozymes or RNAi compositionstargeting a VEGF nucleic acid; anti-VEGF aptamers, anti-VEGF antibodiesor soluble VEGF receptor decoys that prevent binding of a VEGF to itscognate receptor; antisense, ribozymes, or RNAi compositions targeting acognate VEGF receptor (VEGFR) nucleic acid; anti-VEGFR aptamers oranti-VEGFR antibodies that bind to a cognate VEGFR receptor; and VEGFRtyrosine kinase inhibitors.

By “an amount sufficient to suppress a neovascular disorder” is meantthe effective amount of an antagonist, in a combination of theinvention, required to treat or prevent a neovascular disorder orsymptom thereof. The “effective amount” of active antagonists used topractice the present invention for therapeutic treatment of conditionscaused by or contributing to the neovascular disorder varies dependingupon the manner of administration, anatomical location of theneovascular disorder, the age, body weight, and general health of thepatient. Ultimately, a physician or veterinarian will decide theappropriate amount and dosage regimen. Such amount is referred to as anamount sufficient to suppress a neovascular disorder.

Other features and advantages of the invention will be apparent from thefollowing detailed description, and from the claims.

A “variant” of polypeptide X refers to a polypeptide having the aminoacid sequence of peptide X in which is altered in one or more amino acidresidues. The variant may have “conservative” changes, wherein asubstituted amino acid has similar structural or chemical properties(e.g., replacement of leucine,with isoleucine). More rarely, a variantmay have “nonconservative” changes (e.g., replacement of glycine withtryptophan). Analogous minor variations may also include amino aciddeletions or insertions, or both. Guidance in determining which aminoacid residues may be substituted, inserted, or deleted withoutabolishing biological or immunological activity may be found usingcomputer programs well known in the art, for example, LASERGENE software(DNASTAR).

The term “variant,” when used in the context of a polynucleotidesequence, may encompass a polynucleotide sequence related to that ofgene or the coding sequence thereof. This definition may also include,for example, “allelic,” “splice,” “species,” or “polymorphic” variants.A splice variant may have significant identity to a reference molecule,but will generally have a greater or lesser number of polynucleotidesdue to alternative splicing of exons during mRNA processing. Thecorresponding polypeptide may possess additional functional domains oran absence of domains. Species variants are polynucleotide sequencesthat vary from one species to another. The resulting polypeptidesgenerally will have significant amino acid identity relative to eachother. A polymorphic variant is a variation in the polynucleotidesequence of a particular gene between individuals of a given species.

The term “vector” refers to a nucleic acid molecule capable oftransporting another nucleic acid to which it has been linked. One typeof useful vector is an episome, i.e., a nucleic acid capable ofextra-chromosomal replication. Useful vectors are those capable ofautonomous replication and/or expression of nucleic acids to which theyare linked. Vectors capable of directing the expression of genes towhich they are operatively linked are referred to herein as “expressionvectors”. In general, expression vectors of utility in recombinant DNAtechniques are often in the form of “plasmids” which refer generally tocircular double stranded DNA loops which, in their vector form are notbound to the chromosome. In the present specification, “plasmid” and“vector” are used interchangeably as the plasmid is the most commonlyused form of vector. However, the invention is intended to include suchother forms of expression vectors which serve equivalent functions andwhich become known in the art subsequently hereto.

Combination Therapy

The invention is based, in part, upon the specific inhibition of bothVEGF and PDGF activities using appropriate growth factor antagonists asa potent treatment for patients having a neovascular disorder. Theadministration of a combination of a PDGF antagonist and a VEGFantagonist affords greater therapeutic benefits for treating an ocularneovascular disorder than either antagonist administered alone. Thecombined action of anti-VEGF and anti-PDGF agents is unexpected in lightof studies evidencing no apparent cooperation between the two factors instimulating angiogenesis in a retinal endothelial cell system (seeCastellon et al., (2001) Exp. Eye Res. 74: 523-35).

PDGF and VEGF are important stimuli for the growth of new blood vesselsthroughout the body, especially in the eye. Combination therapy directedat inhibiting both PDGF and VEGF biological activities provides a methodfor treating or preventing the neovascular disorder.

Accordingly, the invention features methods and compositions forsuppressing a neovascular disorder using combination therapy. Inparticular, the present invention utilizes two distinct intercellularcommunication signaling pathways operative in vascular cells, namelyPDGF and VEGF signaling, as therapeutic targets in the treatment of aneovascular disorder, such as an ocular neovascular disorder. Thiscombination method is especially useful for treating any number ofophthamalogical diseases and disorders marked by the development ofocular neovascularization, including, but not limited to, optic discneovascularization, iris neovascularization, retinal neovascularization,choroidal neovascularization, corneal neovascularization, vitrealneovascularization, glaucoma, pannus, pterygium, macular edema, diabeticmacular edema, vascular retinopathy, retinal degeneration, maculardegeneration, uveitis, inflammatory diseases of the retina, andproliferative vitreoretinopathy. The combination therapy, consisting ofantagonists that inhibit PDGF (such as PDGF-B) and VEGF (such as VEGF-A)signaling results in an increased treatment efficacy compared to eitherof the two therapies being used independently. While the examplesdiscussed below describe the combination of a single PDGF antagonist anda single VEGF antagonist, it is understood that the combination ofmultiple antagonistic agents may be desirable.

Anti-PDGF and anti-VEGF combination therapy according to the inventionmay be performed alone or in conjunction with another therapy and may beprovided at home, the doctor's office, a clinic, a hospital's outpatientdepartment, or a hospital. Treatment generally begins at a hospital sothat the doctor can observe the therapy's effects closely and make anyadjustments that are needed. The duration of the combination therapydepends on the type of neovascular disorder being treated, the age andcondition of the patient, the stage and type of the patient's disease,and how the patient responds to the treatment. Additionally, a personhaving a greater risk of developing a neovascular disorder (e.g., adiabetic patient) may receive treatment to inhibit or delay the onset ofsymptoms. One significant advantage provided by the present invention isthat the combination of a PDGF antagonist and a VEGF antagonist for thetreatment of a neovascular disorder allows for the administration of alow dose of each antagonist and less total active antagonist, thusproviding similar efficacy with less toxicity and side effects, andreduced costs.

The dosage and frequency of administration of each component of thecombination can be controlled independently. For example, one antagonistmay be administered three times per day, while the second antagonist maybe administered once per day. Combination therapy may be given inon-and-off cycles that include rest periods so that the patient's bodyhas a chance to recover from any as yet unforeseen side-effects. Theantagonists may also be formulated together such that one administrationdelivers both antagonists.

PDGF and VEGF Antagonist Targets

PDGF was originally isolated from platelet lysates and identified as themajor growth-promoting activity present in serum but not in plasma. Themitogenic activity of PDGF was first shown to act on connective tissuecells, such as fibroblasts and smooth muscle cells, and in glial cellsin culture. Two homologous PDGF isoforms have been identified, PDGF Aand B, which are encoded by separate genes (on chromosomes 7 and 22).The most abundant species from platelets is the AB heterodimer, althoughall three possible dimers (AA, AB and BB) occur naturally. Followingtranslation, PDGF dimers are processed into approximately 30 kDasecreted proteins.

Two cell surface proteins that bind PDGF with high affinity have beenidentified, alpha. and beta. (Heldin et al., (1981) Proc. Natl. Acad.Sci. (USA) 78: 3664; Williams et al., (1981) Proc. Natl. Acad. Sci.(USA) 79: 5867). Both species contain five immunoglobulin-likeextracellular domains, a single transmembrane domain and anintracellular tyrosine kinase domain separated by a kinase insertdomain. In the last several years, the specificities of the three PDGFisoforms for the three receptor dimers (alpha/alpha, alpha/beta, andbeta/beta.) have been elucidated. The alpha-receptor homodimer binds allthree PDGF isoforms with high affinity, the beta-receptor homodimerbinds only PDGF BB with high affinity and PDGF AB with approximately10-fold lower affinity, and the alpha/beta.-receptor heterodimer bindsPDGF BB and PDGF AB with high affinity (Westermark & Heldin (1993) ActaOncologica 32:101). The specificity pattern appears to result from theability of the A-chain to bind only to the alpha-receptor and of theB-chain to bind to both alpha and beta-receptor subunits with highaffinity.

In general, the invention provides for agents that inhibit one or morePDGF activities. These PDGF-inhibitory agents, or PDGF antagonists mayact on one or more forms of the PDGF ligand. Platelet derived growthfactors includes homo- or heterodimers of A-chain (PDGF-A) and B-chain(PDGF-B) that exert their action via binding to and dimerization of tworelated receptor tyrosine kinases, [alpha]-receptors (PDGFR-[alpha]) and[beta]-receptors (PDGFR-[beta]). In addition, PDGF-C and PDGF-D, two newprotease-activated ligands for the PDGFR complexes, have been identified(see Li et al., (2000) Nat. Cell. Biol. 2: 302-9; Bergsten et al.,(2001) Nat. Cell. Biol. 3: 512-6; and Uutele et al., (2001) Circulation103: 2242-47). Due to the different ligand binding specificities of thePDGFRs it is known that PDGFR-[alpha][alpha] binds PDGF-AA, PDGF-BB,PDGF-AB, and PDGF-CC; PDGFR-[beta][beta] binds PDGF-BB and PDGF-DD;whereas PDGFR-[alpha][beta] binds PDGF-AB, PDGF-BB, PDGF-CC, and PDGF-DD(see Betsholtz et al., (2001) BioEssays 23: 494-507).

VEGF is a secreted disulfide-linked homodimer that selectivelystimulates endothelial cells to proliferate, migrate, and producematrix-degrading enzymes (Conn et al., (1990) Proc. Natl. Acad. Sci.(USA) 87:1323-1327); Ferrara and Henzel (1989) Biochem. Biophys. Res.Commun.161: 851-858); Pepper et al., (1991) Biochem. Biophys. Res.Commun. 181:902-906; Unemori et al., (1992) J. Cell. Physiol.153:557-562), all of which are processes required for the formation ofnew vessels. VEGF occurs in four forms (VEGF-121, VEGF-165, VEGF-189,VEGF-206) as a result of alternative splicing of the VEGF gene (Houck etal., (1991) Mol. Endocrinol. 5:1806-1814; Tischer et al., (1991) J.Biol. Chem. 266:11947-11954). The two smaller forms are diffusiblewhereas the larger two forms remain predominantly localized to the cellmembrane as a consequence of their high affinity for heparin. VEGF-165also binds to heparin and is the most abundant form. VEGF-121, the onlyform that does not bind to heparin, appears to have a lower affinity forVEGF receptors (Gitay-Goren et al., (1996) J. Biol. Chem. 271:5519-5523)as well as lower mitogenic potency (Keyt et al., (1996) J. Biol. Chem.271:7788-7795). The biological effects of VEGF are mediated by twotyrosine kinase receptors (Flt-1 and Flk-1/KDR) whose expression ishighly restricted to cells of endothelial origin (de Vries et al.,(1992) Science 255:989-991; Millauer et al., (1993) Cell 72:835-846;Terman et al., (1991) Oncogene 6:519-524). While the expression of bothfunctional receptors is required for high affinity binding, thechemotactic and mitogenic signaling in endothelial cells appears tooccur primarily through the KDR receptor (Park et al., (1994) J. Biol.Chem. 269:25646-25654; Seetharam et al., (1995) Oncogene 10:135-147;Waltenberger et al., (1994) J. Biol. Chem. 26988-26995). The importanceof VEGF and VEGF receptors for the development of blood vessels hasrecently been demonstrated in mice lacking a single allele for the VEGFgene (Carmneliet et al., (1996) Nature 380:435-439; Ferrara et al.,(1996) Nature 380:439-442) or both alleles of the Flt-1 (Fong et al.,(1995) Nature 376:66-70) or Flk-1 genes (Shalaby et al., (1995) Nature376:62-66). In each case, distinct abnormalities in vessel formationwere observed resulting in embryonic lethality.

Compensatory angiogenesis induced by tissue hypoxia is now known to bemediated by VEGF (Levy et al., (1996) J. Biol. Chem. 2746-2753); Shweikiet al., (1992) Nature 359:843-845). Studies in humans have shown thathigh concentrations of VEGF are present in the vitreous in angiogenicretinal disorders but not in inactive or non-neovascularization diseasestates. Human choroidal tissue excised after experimental submacularsurgery have also shown high VEGF levels.

In addition to being the only known endothelial cell specific mitogen,VEGF is unique among angiogenic growth factors in its ability to inducea transient increase in blood vessel permeability to macromolecules(hence its original and alternative name, vascular permeability factor,VPF) (see Dvorak et al., (1979) J. Immunol. 122:166-174; Senger et al.,(1983) Science 219:983-985; Senger et al., (1986) Cancer Res.46:5629-5632). Increased vascular permeability and the resultingdeposition of plasma proteins in the extravascular space assists the newvessel formation by providing a provisional matrix for the migration ofendothelial cells (Dvorak et al., (1995) Am. J. Pathol. 146:1029-1039).Hyperpermeability is indeed a characteristic feature of new vessels,including those associated with tumors.

PDGF and VEGF Antagonists

General

The invention provides antagonists (i.e., inhibitors) of PDGF and VEGFfor use together in combination therapy for neovascular disorders.Specific PDGF antagonists and VEGF antagonists are known in the art andare described briefly in the sections that follow. Still other PDGFantagonists and VEGF antagonists that are now, or that have become,available to the skilled artisan include the antibodies, aptamers,antisense oligomers, ribozymes, and RNAi compositions that may beidentified and produced using practices that are routine in the art inconjunction with the teachings and guidance of the specification,including the further-provided sections appearing below.

PDGF Antagonists

Generally, inhibition of PDGF (for example, PDGF-B) may be accomplishedin a variety of ways. For example, a variety of PDGF antagonists thatinhibit the activity or production of PDGF are available and can be usedin the methods of the present invention. Exemplary PDGF antagonistsinclude nucleic acid ligands or aptamers of PDGF, such as thosedescribed below. Alternatively, the PDGF antagonist may be, for example,an anti-PDGF antibody or antibody fragment. Accordingly, the PDGFmolecule is rendered inactive by inhibiting its binding to a receptor.In addition, nucleic acid molecules such as antisense RNA, ribozymes,and RNAi molecules that inhibit PDGF expression at the nucleic acidlevel are useful as antagonists in the invention. Other PDGF antagonistsinclude peptides, proteins, cyclic peptides, or small organic compounds.Furthermore, the signaling activity of PDGF may be inhibited bydisrupting its downstream signaling, for example, by using a number ofsmall molecule tyrosine kinase inhibitory antagonists including thosedescribed below. The ability of a compound or agent to serve as a PDGFantagonist may be determined according to the methods known in art and,further, as set forth in, e.g., Dai et al., (2001) Genes & Dev. 15:1913-25; Zippel, et al., (1989) Eur. J. Cell Biol. 50(2):428-34; andZwiller, et al., (1991) Oncogene 6: 219-21.

The invention further includes PDGF antagonists known in the art as wellas those supported below and any and all equivalents that are within thescope of ordinary skill to create. For example, inhibitory antibodiesdirected against PDGF are known in the art, e.g., those described inU.S. Pat. Nos. 5,976,534, 5,833,986, 5,817,310, 5,882,644, 5,662,904,5,620,687, 5,468,468, and PCT WO 2003/025019, the contents of which areincorporated by reference in their entirety. In addition, the inventioninclude N-phenyl-2-pyrimidine-amine derivatives that are PDGFantagonists, such as those disclosed in U.S. Pat. No. 5,521,184, as wellas WO2003/013541, WO2003/078404, WO2003/099771, WO2003/015282, andWO2004/05282 which are hereby incorporated in their entirety byreference.

Small molecules that block the action of PDGF are known in the art,e.g., those described in U.S. Pat. No. 6,528,526 (PDGFR tyrosine kinaseinhibitors), U.S. Pat. No. 6,524,347 (PDGFR tyrosine kinase inhibitors),U.S. Pat. No. 6,482,834 (PDGFR tyrosine kinase inhibitors), U.S. Pat.No. 6,472,391 (PDGFR tyrosine kinase inhibitors), U.S. Pat. Nos.6,696,434, 6,331,555, 6,251,905, 6,245,760, 6,207,667, 5,990,141,5,700,822, 5,618,837 and 5,731,326, the contents of which areincorporated by reference in their entirety.

Proteins and polypeptides that block the action of PDGF are known in theart, e.g., those described in U.S. Pat. No. 6,350,731 (PDGF peptideanalogs), U.S. Pat. No. 5,952,304, the contents of which areincorporated by reference in their entirety.

Bis mono- and bicyclic aryl and heteroaryl compounds which inhibit EGFand/or PDGF receptor tyrosine kinase are known in the art, e.g., thosedescribed in, e.g U.S. Pat. Nos. 5,476,851, 5,480,883, 5,656,643,5,795,889, and 6,057,320, the contents of which are incorporated byreference in their entirety.

Antisense oligonucleotides for the inhibition of PDGF are known in theart, e.g., those described in U.S. Pat. Nos. 5,869,462, and 5,821,234,the contents of each of which are incorporated by reference in theirentirety.

Aptamers (also known as nucleic acid ligands) for the inhibition of PDGFare known in the art, e.g., those described in, e.g., U.S. Pat. Nos.6,582,918, 6,229,002, 6,207,816, 5,668,264, 5,674,685, and 5,723,594,the contents of each of which are incorporated by reference in theirentirety.

Other compounds for inhibiting PDGF known in the art include thosedescribed in U.S. Pat. Nos. 5,238,950, 5,418,135, 5,674,892, 5,693,610,5,700,822, 5,700,823, 5,728,726, 5,795,910, 5,817,310, 5,872,218,5,932,580, 5,932,602, 5,958,959, 5,990,141, 6,358,954, 6,537,988 and6,673,798, the contents of each of which are incorporated by referencein their entirety.

VEGF Antagonists

Inhibition of VEGF (for example, VEGF-A) is accomplished in a variety ofways. For example, a variety of VEGF antagonists that inhibit theactivity or production of VEGF, including nucleic acid molecules such asaptamers, antisense RNA, ribozymes, RNAi molecules, and VEGF antibodies,are available and can be used in the methods of the present invention.Exemplary VEGF antagonists include nucleic acid ligands or aptamers ofVEGF, such as those described below. A particularly useful antagonist toVEGF-A is EYE001 (previously referred to as NX1838), which is amodified, PEGylated aptamer that binds with high and specific affinityto the major soluble human VEGF isoform (see, U.S. Pat. Nos. 6,011,020;6,051,698; and 6,147,204). The aptamer binds and inactivates VEGF in amanner similar to that of a high-affinity antibody directed towardsVEGF. Another useful VEGF aptamer is EYE001 in its non-pegylated form.Alternatively, the VEGF antagonist may be, for example, an anti-VEGFantibody or antibody fragment. Accordingly, the VEGF molecule isrendered inactive by inhibiting its binding to a receptor. In addition,nucleic acid molecules such as antisense RNA, ribozymes, and RNAimolecules that inhibit VEGF expression or RNA stability at the nucleicacid level are useful antagonists in the methods and compositions of theinvention. Other VEGF antagonists include peptides, proteins, cyclicpeptides, and small organic compound. For example, soluble truncatedforms of VEGF that bind to the VEGF receptor without concomitantsignaling activity also serve as antagonists. Furthermore, the signalingactivity of VEGF may be inhibited by disrupting its downstreamsignaling, for example, by using a number of antagonists including smallmolecule inhibitors of a VEGF receptor tyrosine kinase activity, asdescribed further below.

The ability of a compound or agent to serve as a VEGF antagonist may bedetermined according to any number of standard methods well known in theart. For example, one of the biological activities of VEGF is toincrease vascular permeability through specific binding to receptors onvascular endothelial cells. The interaction results in relaxation of thetight endothelial junctions with subsequent leakage of vascular fluid.Vascular leakage induced by VEGF can be measured in vivo by followingthe leakage of Evans Blue Dye from the vasculature of the guinea pig asa consequence of an intradermal injection of VEGF (Dvorak et al., inVascular Permeability Factor/Vascular Endothelial Growth FactorMicrovascular Hyperpermeability and Angiogenesis; and (1995) Am. J.Pathol. 146:1029). Similarly, the assay can be used to measure theability of an antagonist to block this biological activity of VEGF.

In one useful example of a vascular permeability assay, VEGF₁₆₅ (20-30nM) is premixed ex vivo with EYE001 (30 nM to 1 μM) or a candidate VEGFantagonist and subsequently administered by intradermal injection intothe shaved skin on the dorsum of guinea pigs. Thirty minutes followinginjection, the Evans Blue dye leakage around the injection sites isquantified according to standard methods by use of a computerizedmorphometric analysis system. A compound that inhibits VEGF-inducedleakage of the indicator dye from the vasculature is taken as being auseful antagonist in the methods and compositions of the invention.

Another assay for determining whether a compound is a VEGF antagonist isthe so-called corneal angiogenesis assay. In this assay, methacyratepolymer pellets containing VEGF₁₆₅ (3 pmol) are implanted into thecorneal stroma of rats to induce blood vessel growth into the normallyavascular cornea. A candidate VEGF antagonist is then administeredintravenously to the rats at doses of 1 mg/kg, 3 mg/kg, and 10 mg/kgeither once or twice daily for 5 days. At the end of the treatmentperiod, all of the individual corneas are photomicrographed. The extentto which new blood vessels develop in the corneal tissue, and theirinhibition by the candidate compound, are then quantified bystandardized morphometric analysis of the photomicrographs. A compoundthat inhibits VEGF-dependent angiogenesis in the cornea when compared totreatment with phosphate buffered saline (PBS) is taken as being auseful antagonist in the methods and compositions of the invention.

Candidate VEGF antagonists are also identified using the mouse model ofretinopathy of prematurity. In one useful example, litters of 9, 8, 8,7, and 7 mice, respectively, are left in room air or made hyperoxic andare treated intraperitoneally with phosphate buffered saline (PBS) or acandidate VEGF antagonist (for example, at 1 mg/kg, 3 mg/kg, or 10mg/kg/day). The endpoint of the assay, outgrowth of new capillariesthrough the inner limiting membrane of the retina into the vitreoushumor, is then assessed by microscopic identification and counting ofthe neovascular buds in 20 histologic sections of each eye from all ofthe treated and control mice. A reduction in retinal neovasculature inthe treated mice relative to the untreated control is taken asidentifying a useful VEGF antagonist.

In still another exemplary screening assay, candidate VEGF antagonistsare identified using an in vivo human tumor xenograft assay. In thisscreening assay, in vivo efficacy of a candidate VEGF antagonist istested in human tumor xenografts (A673 rhabdomyosarcoma and Wilms tumor)implanted in nude mice. Mice are then treated with the candidate VEGFantagonist (e.g., 10 mg/kg given intraperitoneally once a day followingdevelopment of established tumors (200 mg)). Control groups are treatedwith a control agent. Candidate compounds identified as inhibiting A673rhabdomyosarcoma tumor growth and Wilms tumor relative to the controlare taken as being useful antagonists in the methods and compositions ofthe invention.

Additional methods of assaying for a VEGF antagonist activity are knownin the art and described in further detail below.

The invention further includes VEGF antagonists known in the art as wellas those supported below and any and all equivalents that are within thescope of ordinary skill to create. For example, inhibitory antibodiesdirected against VEGF are known in the art, e.g., those described inU.S. Pat. Nos. 6,524,583, 6,451,764 (VRP antibodies), U.S. Pat. Nos.6,448,077, 6,416,758, 6,403,088 (to VEGF-C), U.S. Pat. No. 6,383,484 (toVEGF-D), U.S. Pat. No. 6,342,221 (anti-VEGF antibodies), U.S. Pat. Nos.6,342,219 6,331,301 (VEGF-B antibodies), and U.S. Pat. No. 5,730,977,and PCT publications WO96/30046, WO 97/44453, and WO 98/45331, thecontents of which are incorporated by reference in their entirety.

Antibodies to VEGF receptors are also known in the art, such as thosedescribed in, for example, U.S. Pat. Nos. 5,840,301, 5,874,542,5,955,311, 6,365,157, and PCT publication WO 04/003211, the contents ofwhich are incorporated by reference in their entirety.

Small molecules that block the action of VEGF by, e.g., inhibiting aVEGFR-associated tyrosine kinase activity, are known in the art, e.g.,those described in U.S. Pat. Nos. 6,514,971, 6,448,277 , 6,414,148,6,362,336, 6,291,455, 6,284,751, 6,177,401, 6,071,921, and 6,001,885(retinoid inhibitors of VEGF expression), the contents of each of whichare incorporated by reference in their entirety.

Proteins and polypeptides that block the action of VEGF are known in theart, e.g., those described in U.S. Pat. Nos. 6,576,608, 6,559,126,6,541,008, 6,515,105, 6,383,486 (VEGF decoy receptor), U.S. Pat. No.6,375,929 (VEGF decoy receptor), U.S. Pat. No. 6,361,946 (VEFG peptideanalog inhibitors), U.S. Pat. No. 6,348,333 (VEGF decoy receptor), U.S.Pat. No. 6,559,126 (polypeptides that bind VEGF and block binding toVEGFR), U.S. Pat. No. 6,100,071 (VEGF decoy receptor), and U.S. Pat. No.5,952,199, the contents of each of which are incorporated by referencein their entirety.

Short interfering nucleic acids (siNA), short interfering RNA (siRNA),double stranded RNA (dsRNA), microRNA (miRNA) and short hairpin RNA(shRNA) capable of mediating RNA interference (RNAi) against VEGF and/orVEGFR gene expression and/or activity are known in the art, for example,as disclosed in PCT publication WO 03/070910, the contents of which isincorporated by reference in its entirety.

Antisense oligonucleotides for the inhibition of VEGF are known in theart, e.g., those described in, e.g., U.S. Pat. Nos. 5,611,135,5,814,620, 6,399,586, 6,410,322, and 6,291,667, the contents of each ofwhich are incorporated by reference in their entirety.

Aptamers (also known as nucleic acid ligands) for the inhibition of VEGFare known in the art, e.g., those described in, e.g., U.S. Pat. Nos.6,762,290, 6,426,335, 6,168,778, 6,051,698, and 5,859,228, the contentsof each of which are incorporated by reference in their entirety.

Antibody Antagonists

The invention includes antagonist antibodies directed against PDGF andVEGF as well as their cognate receptors PDGFR and VEGFR. The antibodyantagonists of the invention block binding of a ligand with its cognatereceptor. Accordingly, a PDGF antagonist antibody of the inventionincludes antibodies directed against a PDGF as well as a PDGFR target.

The antagonist antibodies of the invention include monoclonal inhibitoryantibodies. Monoclonal antibodies, or fragments thereof, encompass allimmunoglobulin classes such as IgM, IgG, IgD, IgE, IgA, or theirsubclasses, such as the IgG subclasses or mixtures thereof. IgG and itssubclasses are useful, such as IgG₁, IgG₂, IgG_(2a), IgG_(2b), IgG₃ orIgG_(M). The IgG subtypes IgG_(1/kappa) and IgG_(2b/kapp) are includedas useful embodiments. Fragments which may be mentioned are alltruncated or modified antibody fragments with one or twoantigen-complementary binding sites which show high binding andneutralizing activity toward mammalian PDGF or VEGF (or their cognatereceptors), such as parts of antibodies having a binding site whichcorresponds to the antibody and is formed by light and heavy chains,such as Fv, Fab or F(ab′)₂ fragments, or single-stranded fragments.Truncated double-stranded fragments such as Fv, Fab or F(ab′)₂ areparticularly useful. These fragments can be obtained, for example, byenzymatic means by eliminating the Fc part of the antibody with enzymessuch as papain or pepsin, by chemical oxidation or by geneticmanipulation of the antibody genes. It is also possible and advantageousto use genetically manipulated, non-truncated fragments. The anti-PDGFor VEGF antibodies or fragments thereof can be used alone or inmixtures.

The novel antibodies, antibody fragments, mixtures or derivativesthereof advantageously have a binding affinity for PDGF or VEGF (ortheir cognate receptors) in a range from 1×10⁻⁷ M to 1×10⁻¹² M, or from1×10⁻⁸ M to 1×10⁻¹¹ M, or from 1×10⁻⁹ M to 5×10⁻¹⁰ M.

The antibody genes for the genetic manipulations can be isolated, forexample from hybridoma cells, in a manner known to the skilled worker.For this purpose, antibody-producing cells are cultured and, when theoptical density of the cells is sufficient, the mRNA is isolated fromthe cells in a known manner by lysing the cells with guanidiniumthiocyanate, acidifying with sodium acetate, extracting with phenol,chloroform/isoamyl alcohol, precipitating with isopropanol and washingwith ethanol. cDNA is then synthesized from the mRNA using reversetranscriptase. The synthesized cDNA can be inserted, directly or aftergenetic manipulation, for example, by site-directed mutagenesis,introduction of insertions, inversions, deletions, or base exchanges,into suitable animal, fungal, bacterial or viral vectors and beexpressed in appropriate host organisms. Useful bacterial or yeastvectors are pBR322, pUC18/19, pACYC184, lambda or yeast mu vectors forthe cloning of the genes and expression in bacteria such as E. coli orin yeasts such as Saccharomyces cerevisiae.

The invention furthermore relates to cells that synthesize PDGF or VEGFantibodies. These include animal, fungal, bacterial cells or yeast cellsafter transformation as mentioned above. They are advantageouslyhybridoma cells or trioma cells, typically hybridoma cells. Thesehybridoma cells can be produced, for example, in a known manner fromanimals immunized with PDGF or VEGF (or their cognate receptors) andisolation of their antibody-producing B cells, selecting these cells forPDGF or VEGF-binding antibodies and subsequently fusing these cells to,for example, human or animal, for example, mouse myeloma cells, humanlymphoblastoid cells or heterohybridoma cells (see, e.g., Koehler etal., (1975) Nature 256: 496) or by infecting these cells withappropriate viruses to produce immortalized cell lines. Hybridoma celllines produced by fusion are useful and mouse hybridoma cell lines areparticularly useful. The hybridoma cell lines of the invention secreteuseful antibodies of the IgG type. The binding of the mAb antibodies ofthe invention bind with high affinity and reduce or neutralize thebiological (e.g., angiogenic) activity of PDGF or VEGF.

The invention further includes derivatives of these anti-PDGF or VEGFantibodies which retain their PDGF or VEGF-inhibiting activity whilealtering one or more other properties related to their use as apharmaceutical agent, e.g., serum stability or efficiency of production.Examples of such anti-PDGF or VEGF antibody derivatives includepeptides, peptidomimetics derived from the antigen-binding regions ofthe antibodies, and antibodies, antibody fragments or peptides bound tosolid or liquid carriers such as polyethylene glycol, glass, syntheticpolymers such as polyacrylamide, polystyrene, polypropylene,polyethylene or natural polymers such as cellulose, Sepharose oragarose, or conjugates with enzymes, toxins or radioactive ornonradioactive markers such as ³H, ¹²³I, ¹²⁵I, ¹³¹I, ³²P, ³⁵S, ¹⁴C,⁵¹Cr, ³⁶Cl, ⁵⁷Co, ⁵⁵Fe, ⁵⁹Fe, ⁹⁰Y, ^(99m)Tc, ⁷⁵Se, or antibodies,fragments, or peptides covalently bonded to fluorescent/chemiluminescentlabels such as rhodamine, fluorescein, isothiocyanate, phycoerythrin,phycocyanin, fluorescamine, metal chelates, avidin, streptavidin orbiotin.

The novel antibodies, antibody fragments, mixtures, and derivativesthereof can be used directly, after drying, for example freeze drying,after attachment to the abovementioned carriers or after formulationwith other pharmaceutical active and ancillary substances for producingpharmaceutical preparations. Examples of active and ancillary substanceswhich may be mentioned are other antibodies, antimicrobial activesubstances with a microbiocidal or microbiostatic action such asantibiotics in general or sulfonamides, antitumor agents, water,buffers, salines, alcohols, fats, waxes, inert vehicles or othersubstances customary for parenteral products, such as amino acids,thickeners or sugars. These pharmaceutical preparations are used tocontrol diseases, and are useful to control ocular neovascular disordersand diseases including AMD and diabetic retinopathy.

The novel antibodies, antibody fragments, mixtures or derivativesthereof can be used in therapy or diagnosis directly or after couplingto solid or liquid carriers, enzymes, toxins, radioactive ornonradioactive labels or to fluorescent/chemiluminescent labels asdescribed above.

The human PDGF or VEGF monoclonal antibodies of the present inventionmay be obtained by any means known in the art. For example, a mammal isimmunized with human PDGF or VEGF (or their cognate receptors). Purifiedhuman PDGF and VEGF is commercially available (e.g., from Cell Sciences,Norwood, Mass., as well as other commercial vendors). Alternatively,human PDGF or VEGF (or their cognate receptors) may be readily purifiedfrom human placental tissue. The mammal used for raising anti-human PDGFor VEGF antibody is not restricted and may be a primate, a rodent (suchas mouse, rat or rabbit), bovine, sheep, goat or dog.

Next, antibody-producing cells such as spleen cells are removed from theimmunized animal and are fused with myeloma cells. The myeloma cells arewell-known in the art (e.g., p3x63-Ag8-653, NS-0, NS-1 or P3U1 cells maybe used). The cell fusion operation may be carried out by anyconventional method known in the art.

The cells, after being subjected to the cell fusion operation, are thencultured in HAT selection medium so as to select hybridomas. Hybridomaswhich produce antihuman monoclonal antibodies are then screened. Thisscreening may be carried out by, for example, sandwich enzyme-linkedimmunosorbent assay (ELISA) or the like in which the produced monoclonalantibodies are bound to the wells to which human PDGF or VEGF (or theircognate receptor) is immobilized. In this case, as the secondaryantibody, an antibody specific to the immunoglobulin of the immunizedanimal, which is labeled with an enzyme such as peroxidase, alkalinephosphatase, glucose oxidase, beta-D-galactosidase, or the like, may beemployed. The label may be detected by reacting the labeling enzyme withits substrate and measuring the generated color. As the substrate,3,3-diaminobenzidine, 2,2-diaminobis-o-dianisidine, 4-chloronaphthol,4-aminoantipyrine, o-phenylenediamine or the like may be produced.

By the above-described operation, hybridomas which produce anti-humanPDGF or VEGF antibodies can be selected. The selected hybridomas arethen cloned by the conventional limiting dilution method or soft agarmethod. If desired, the cloned hybridomas may be cultured on a largescale using a serum-containing or a serum free medium, or may beinoculated into the abdominal cavity of mice and recovered from ascites,thereby a large number of the cloned hybridomas may be obtained.

From among the selected anti-human PDGF or VEGF monoclonal antibodies,those that have an ability to prevent binding and activation of thecorresponding ligand/receptor pair (e.g., in a cell-based PDGF or VEGFassay system (see above)) are then chosen for further analysis andmanipulation. If the antibody blocks receptor/ligand binding and/oractivation, it means that the monoclonal antibody tested has an abilityto reduce or neutralize the PDGF or VEGF activity of human PDGF or VEGF.That is, the monoclonal antibody specifically recognizes and/orinterferes with the critical binding site of human PDGF or VEGF (ortheir cognate receptors).

The monoclonal antibodies herein further include hybrid and recombinantantibodies produced by splicing a variable (including hypervariable)domain of an anti-PDGF or VEGF antibody with a constant domain (e.g.,“humanized” antibodies), or a light chain with a heavy chain, or a chainfrom one species with a chain from another species, or fusions withheterologous proteins, regardless of species of origin or immunoglobulinclass or subclass designation, as well as antibody fragments [e.g., Fab,F(ab)₂, and Fv], so long as they exhibit the desired biologicalactivity. [See, e.g., U.S. Pat. No. 4,816,567 and Mage & Lamoyi, inMonoclonal Antibody Production Techniques and Applications, pp. 79-97(Marcel Dekker, Inc.), New York (1987)].

Thus, the term “monoclonal” indicates that the character of the antibodyobtained is from a substantially homogeneous population of antibodies,and is not to be construed as requiring production of the antibody byany particular method. For example, the monoclonal antibodies to be usedin accordance with the present invention may be made by the hybridomamethod first described by Kohler & Milstein, Nature 256:495 (1975), ormay be made by recombinant DNA methods (U.S. Pat. No. 4,816,567). The“monoclonal antibodies” may also be isolated from phage librariesgenerated using the techniques described in McCafferty et al., Nature348:552-554 (1990), for example.

“Humanized” forms of non-human (e.g., murine) antibodies are specificchimeric immunoglobulins, immunoglobulin chains or fragments thereof(such as Fv, Fab, Fab′, F(ab)₂ or other antigen-binding subsequences ofantibodies) which contain minimal sequence derived from non-humanimmunoglobulin. For the most part, humanized antibodies are humanimmunoglobulins (recipient antibody) in which residues from thecomplementary determining regions (CDRs) of the recipient antibody arereplaced by residues from the CDRs of a non-human species (donorantibody) such as mouse, rat or rabbit having the desired specificity,affinity and capacity. In some instances, Fv framework region (FR)residues of the human immunoglobulin are replaced by correspondingnon-human FR residues. Furthermore, the humanized antibody may compriseresidues that are found neither in the recipient antibody nor in theimported CDR or FR sequences. These modifications are made to furtherrefine and optimize antibody performance. In general, the humanizedantibody will comprise substantially all of at least one, and typicallytwo, variable domains, in which all or substantially all of the CDRregions correspond to those of a non-human immunoglobulin and all orsubstantially all of the FR residues are those of a human immunoglobulinconsensus sequence. The humanized antibody optimally also will compriseat least a portion of an immunoglobulin constant region (Fc), typicallythat of a human immunoglobulin.

Methods for humanizing non-human antibodies are well known in the art.Generally, a humanized antibody has one or more amino acid residuesintroduced into it from a source which is non-human. These non-humanamino acid residues are often referred to as “import” residues, whichare typically taken from an “import” variable domain. Humanization canbe essentially performed following the method of Winter and co-workers(Jones et al., (1986) Nature 321: 522-525; Riechmann et al., (1988)Nature 332: 323-327; and Verhoeyen et al., (1988) Science 239:1534-1536), by substituting rodent CDRs or CDR sequences for thecorresponding sequences of a human antibody. Accordingly, such“humanized” antibodies are chimeric antibodies, wherein substantiallyless than an intact human variable domain has been substituted by thecorresponding sequence from a non-human species. In practice, humanizedantibodies are typically human antibodies in which some CDR residues andpossibly some FR residues are substituted by residues from analogoussites in rodent antibodies.

The choice of human variable domains, both light and heavy, to be usedin making the humanized antibodies is very important to reduceantigenicity. According to the so-called “best-fit” method, the sequenceof the variable domain of a rodent antibody is screened against theentire library of known human variable-domain sequences. The humansequence which is closest to that of the rodent is then accepted as thehuman framework (FR) for the humanized antibody (Sims et al., (1993) J.Immunol., 151:2296; and Chothia and Lesk (1987) J. Mol. Biol., 196:901).Another method uses a particular framework derived from the consensussequence of all human antibodies of a particular subgroup of light orheavy chains. The same framework may be used for several differenthumanized antibodies (Carter et al., (1992) Proc. Natl. Acad. Sci.(USA), 89: 4285; and Presta et al., (1993) J. Immol., 151:2623).

It is further important that antibodies be humanized with retention ofhigh affinity for the antigen and other favorable biological properties.To achieve this goal, according to one useful method, humanizedantibodies are prepared by a process of analysis of the parentalsequences and various conceptual humanized products usingthree-dimensional models of the parental and humanized sequences.Three-dimensional immunoglobulin models are commonly available and arefamiliar to those skilled in the art. Computer programs are availablewhich illustrate and display probable three-dimensional conformationalstructures of selected candidate immunoglobulin sequences. Inspection ofthese displays permits analysis of the likely role of the residues inthe functioning of the candidate immunoglobulin sequence, i.e., theanalysis of residues that influence the ability of the candidateimmunoglobulin to bind its antigen. In this way, FR residues can beselected and combined from the consensus and import sequences so thatthe desired antibody characteristic, such as increased affinity for thetarget antigen(s), is achieved. In general, the CDR residues aredirectly and most substantially involved in influencing antigen binding.

Human monoclonal antibodies directed against PDGF or VEGF are alsoincluded in the invention. Such antibodies can be made by the hybridomamethod. Human myeloma and mouse-human heteromyeloma cell lines for theproduction of human monoclonal antibodies have been described, forexample, by Kozbor (1984) J. Immunol., 133, 3001; Brodeur, et al.,Monoclonal Antibody Production Techniques and Applications, pp. 51-63(Marcel Dekker, Inc., New York, 1987); and Boemer et al., (1991) J.Immunol., 147:86-95.

It is now possible to produce transgenic animals (e.g., mice) that arecapable, upon immunization, of producing a full repertoire of humanantibodies in the absence of endogenous immunoglobulin production. Forexample, it has been described that the homozygous deletion of theantibody heavy-chain joining region (J_(H)) gene in chimeric andgerm-line mutant mice results in complete inhibition of endogenousantibody production. Transfer of the human germ-line immunoglobulin genearray in such gem-line mutant mice will result in the production ofhuman antibodies upon antigen challenge (see, e.g., Jakobovits et al.,(1993) Proc. Natl. Acad. Sci. (USA), 90: 2551; Jakobovits et al., (1993)Nature, 362:255-258; and Bruggermann et al., (1993) Year in Immuno.,7:33).

Alternatively, phage display technology (McCafferty et al., (1990)Nature, 348: 552-553) can be used to produce human antibodies andantibody fragments in vitro, from immunoglobulin variable (V) domaingene repertoires from unimmunized donors (for review see, e.g., Johnsonet al., (1993) Current Opinion in Structural Biology, 3:564-571).Several sources of V-gene segments can be used for phage display. Forexample, Clackson et al., ((1991) Nature, 352: 624-628) isolated adiverse array of anti-oxazolone antibodies from a small randomcombinatorial library of V genes derived from the spleens of immunizedmice. A repertoire of V genes from unimmunized human donors can beconstructed and antibodies to a diverse array of antigens (includingself-antigens) can be isolated essentially following the techniquesdescribed by Marks et al., ((1991) J. Mol. Biol., 222:581-597, orGriffith et al., (1993) EMBO J., 12:725-734).

In a natural immune response, antibody genes accumulate mutations at ahigh rate (somatic hypermutation). Some of the changes introduced willconfer higher affinity, and B cells displaying high-affinity surfaceimmunoglobulin are preferentially replicated and differentiated duringsubsequent antigen challenge. This natural process can be mimicked byemploying the technique known as “chain shuffling” (see Marks et al.,(1992) Bio. Technol., 10:779-783). In this method, the affinity of“primary” human antibodies obtained by phage display can be improved bysequentially replacing the heavy and light chain V region genes withrepertoires of naturally occurring variants (repertoires) of V domaingenes obtained from unimmunized donors. This technique allows theproduction of antibodies and antibody fragments with affinities in thenM range. A strategy for making very large phage antibody repertoireshas been described by Waterhouse et al., ((1993) Nucl. Acids Res.,21:2265-2266).

Gene shuffling can also be used to derive human antibodies from rodentantibodies, where the human antibody has similar affinities andspecificities to the starting rodent antibody. According to this method,which is also referred to as “epitope imprinting”, the heavy or lightchain V domain gene of rodent antibodies obtained by phage displaytechnique is replaced with a repertoire of human V domain genes,creating rodent-human chimeras. Selection on antigen results inisolation of human variable capable of restoring a functionalantigen-binding site, i.e., the epitope governs (imprints) the choice ofpartner. When the process is repeated in order to replace the remainingrodent V domain, a human antibody is obtained (see PCT WO 93/06213,published 1 Apr. 1993). Unlike traditional humanization of rodentantibodies by CDR grafting, this technique provides completely humanantibodies, which have no framework or CDR residues of rodent origin.

Aptamer Antagonists

The invention provides aptamer antagonists directed against PDGF and/orVEGF (or their cognate receptors). Aptamers, also known as nucleic acidligands, are non-naturally occurring nucleic acids that bind to and,generally, antagonize (i.e., inhibit) a pre-selected target.

Aptamers can be made by any known method of producing oligomers oroligonucleotides. Many synthesis methods are known in the art. Forexample, 2′-O-allyl modified oligomers that contain residual purineribonucleotides, and bearing a suitable 3′-terminus such as an invertedthymidine residue (Ortigao et al., Antisense Research and Development,2:129-146 (1992)) or two phosphorothioate linkages at the 3′-terminus toprevent eventual degradation by 3′-exonucleases, can be synthesized bysolid phase beta-cyanoethyl phosphoramidite chemistry (Sinha et al.,Nucleic Acids Res., 12:4539-4557 (1984)) on any commercially availableDNA/RNA synthesizer. One method is the 2′-O-tert-butyldimethylsilyl(TBDMS) protection strategy for the ribonucleotides (Usman et al., J.Am. Chem. Soc., 109:7845-7854 (1987)), and all the required3′-O-phosphoramidites are commercially available. In addition,aminomethylpolystyrene may be used as the support material due to itsadvantageous properties (McCollum and Andrus (1991) Tetrahedron Lett.,32:4069-4072). Fluorescein can be added to the 5′-end of a substrate RNAduring the synthesis by using commercially available fluoresceinphosphoramidites. In general, an aptamer oligomer can be synthesizedusing a standard RNA cycle. Upon completion of the assembly, all baselabile protecting groups are removed by an eight hour treatment at 55°C. with concentrated aqueous ammonia/ethanol (3:1 v/v) in a sealed vial.The ethanol suppresses premature removal of the 2′-O-TBDMS groups thatwould otherwise lead to appreciable strand cleavage at the resultingribonucleotide positions under the basic conditions of the deprotection(Usman et al., (1987) J. Am. Chem. Soc., 109:7845-7854). Afterlyophilization, the TBDMS protected oligomer is treated with a mixtureof triethylamine trihydrofluoride/triethylamine/N-methylpyrrolidinonefor 2 hours at 60° C. to afford fast and efficient removal of the silylprotecting groups under neutral conditions (see Wincott et al., (1995)Nucleic Acids Res., 23:2677-2684). The fully deprotected oligomer canthen be precipitated with butanol according to the procedure of Cathalaand Brunel ((1990) Nucleic Acids Res., 18:201). Purification can beperformed either by denaturing polyacrylamide gel electrophoresis or bya combination of ion-exchange HPLC (Sproat et al., (1995) Nucleosidesand Nucleotides, 14:255-273) and reversed phase HPLC. For use in cells,synthesized oligomers are converted to their sodium salts byprecipitation with sodium perchlorate in acetone. Traces of residualsalts may then be removed using small disposable gel filtration columnsthat are commercially available. As a final step the authenticity of theisolated oligomers may be checked by matrix assisted laser desorptionmass spectrometry (Pieles et al., (1993) Nucleic Acids Res.,21:3191-3196) and by nucleoside base composition analysis.

The disclosed aptamers can also be produced through enzymatic methods,when the nucleotide subunits are available for enzymatic manipulation.For example, the RNA molecules can be made through in vitro RNApolymerase T7 reactions. They can also be made by strains of bacteria orcell lines expressing T7, and then subsequently isolated from thesecells. As discussed below, the disclosed aptamers can also be expressedin cells directly using vectors and promoters.

The aptamers, like other nucleic acid molecules of the invention, mayfurther contain chemically modified nucleotides. One issue to beaddressed in the diagnostic or therapeutic use of nucleic acids is thepotential rapid degration of oligonucleotides in their phosphodiesterform in body fluids by intracellular and extracellular enzymes such asendonucleases and exonucleases before the desired effect is manifest.Certain chemical modifications of the nucleic acid ligand can be made toincrease the in vivo stability of the nucleic acid ligand or to enhanceor to mediate the delivery of the nucleic acid ligand (see, e.g., U.S.Pat. No. 5,660,985, entitled “High Affinity Nucleic Acid LigandsContaining Modified Nucleotides”) which is specifically incorporatedherein by reference.

Modifications of the nucleic acid ligands contemplated in this inventioninclude, but are not limited to, those which provide other chemicalgroups that incorporate additional charge, polarizability,hydrophobicity, hydrogen bonding, electrostatic interaction, andfluxionality to the nucleic acid ligand bases or to the nucleic acidligand as a whole. Such modifications include, but are not limited to,2′-position sugar modifications, 5-position pyrimidine modifications,8-position purine modifications, modifications at exocyclic amines,substitution of 4-thiouridine, substitution of 5-bromo or 5-iodo-uracil;backbone modifications, phosphorothioate or alkyl phosphatemodifications, methylations, unusual base-pairing combinations such asthe isobases isocytidine and isoguanidine and the like. Modificationscan also include 3′ and 5′ modifications such as capping or modificationwith sugar moieties. In some embodiments of the instant invention, thenucleic acid ligands are RNA molecules that are 2′-fluoro (2′-F)modified on the sugar moiety of pyrimidine residues.

The stability of the aptamer can be greatly increased by theintroduction of such modifications and as well as by modifications andsubstitutions along the phosphate backbone of the RNA. In addition, avariety of modifications can be made on the nucleobases themselves whichboth inhibit degradation and which can increase desired nucleotideinteractions or decrease undesired nucleotide interactions. Accordingly,once the sequence of an aptamer is known, modifications or substitutionscan be made by the synthetic procedures described below or by proceduresknown to those of skill in the art.

Other modifications include the incorporation of modified bases (ormodified nucleoside or modified nucleotides) that are variations ofstandard bases, sugars and/or phosphate backbone chemical structuresoccurring in ribonucleic (i.e., A, C, G and U) and deoxyribonucleic(i.e., A, C, G and T) acids. Included within this scope are, forexample: Gm (2′-methoxyguanylic acid), Am (2′-methoxyadenylic acid), Cf(2′-fluorocytidylic acid), Uf (2′-fluorouridylic acid), Ar (riboadenylicacid). The aptamers may also include cytosine or any cytosine-relatedbase including 5-methylcytosine, 4-acetylcytosine, 3-methylcytosine,5-hydroxymethyl cytosine, 2-thiocytosine, 5-halocytosine (e.g.,5-fluorocytosine, 5-bromocytosine, 5-chlorocytosine, and5-iodocytosine), 5-propynyl cytosine, 6-azocytosine,5-trifluoromethylcytosine, N4, N4-ethanocytosine, phenoxazine cytidine,phenothiazine cytidine, carbazole cytidine or pyridoindole cytidine. Theaptamer may further include guanine or any guanine-related baseincluding 6-methylguanine, 1-methylguanine, 2,2-dimethylguanine,2-methylguanine, 7-methylguanine, 2-propylguanine, 6-propylguanine,8-haloguanine (e.g., 8-fluoroguanine, 8-bromoguanine, 8-chloroguanine,and 8-iodoguanine), 8-aminoguanine, 8-sulthydrylguanine,8-thioalkylguanine, 8-hydroxylguanine, 7-methylguanine, 8-azaguanine,7-deazaguanine or 3-deazaguanine. The aptamer may still further includeadenine or any adenine-related base including 6-methyladenine,N6-isopentenyladenine, N6-methyladenine, 1-methyladenine,2-methyladenine, 2-methylthio-N6-isopentenyladenine, 8-haloadenine(e.g., 8-fluoroadenine, 8-bromoadenine, 8-chloroadenine, and8-iodoadenine), 8-aminoadenine, 8-sulfhydryladenine, 8-thioalkyladenine,8-hydroxyladenine, 7-methyladenine, 2-haloadenine (e.g.,2-fluoroadenine, 2-bromoadenine, 2-chloroadenine, and 2-iodoadenine),2-aminoadenine, 8-aaaadenine, 7-deazaadenine or 3-deazaadenine. Alsoincluded are uracil or any uracil-related base including 5-halouracil(e.g., 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil),5-(carboxyhydroxylmethyl)uracil,5-carboxymethylaminomethyl-2-thiouracil,5-carboxymethylaminomethyluracil, dihydrouracil, 1-methylpseudouracil,5-methoxyaminomethyl-2-thiouracil, 5′-methoxycarbonylmethyluracil,5-methoxyuracil, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil,5-methyluracil, uracil-5-oxyacetic acid methylester, uracil-5-oxyaceticacid, pseudouracil, 5-methyl-2-thiouracil, 2-thiouracil,3-(3-amino-3-N-2-carboxypropyl)uracil, 5-methylaminomethyluracil,5-propynyl uracil, 6-azouracil, or 4-thiouracil.

Examples of other modified base variants known in the art include,without limitation, those listed at 37 C.F.R. §1.822(p) (1), e.g.,4-acetylcytidine, 5-(carboxyhydroxylmethyl) uridine, 2′-methoxycytidine,5-carboxymethylaminomethyl-2-thioridine,5-carboxymethylaminomethyluridine, dihydrouridine,2′-O-methylpseudouridine, b-D-galactosylqueosine, inosine,N6-isopentenyladenosine, 1-methyladenosine, 1-methylpseudouridine,1-methylguanosine, 1-methylinosine, 2,2-dimethylguanosine,2-methyladenosine, 2-methylguanosine, 3-methylcytidine,5-methylcytidine, N6-methyladenosine, 7-methylguanosine,5-methylaminomethyluridine, 5-methoxyaminomethyl-2-thiouridine,b-D-mannosylqueosine, 5-methoxycarbonylmethyluridine, 5-methoxyuridine,2-methylthio-N6-isopentenyladenosine,N-((9-b-D-ribofuranosyl-2-methylthiopurine-6-yl)carbamoyl)threonine,N-((9-b-D-ribofuranosylpurine-6-yl)N-methyl-carbamoyl)threonine,urdine-5-oxyacetic acid methylester, uridine-5-oxyacetic acid (v),wybutoxosine, pseudouridine, queosine, 2-thiocytidine,5-methyl-2-thiouridine, 2-thiouridine, 4-thiouridine, 5-methyluridine,N-((9-b-D-ribofuranosylpurine-6-yl)carbamoyl)threonine,2′-O-methyl-5-methyluridine, 2′-O-methyluridine, wybutosine,3-(3-amino-3-carboxypropyl)uridine.

Also included are the modified nucleobases described in U.S. Pat. Nos.3,687,808, 3,687,808, 4,845,205, 5,130,302, 5,134,066, 5,175,273,5,367,066, 5,432,272, 5,457,187, 5,459,255, 5,484,908, 5,502,177,5,525,711, 5,552,540, 5,587,469, 5,594,121, 5,596,091, 5,614,617,5,645,985, 5,830,653, 5,763,588, 6,005,096, and 5,681,941. Examples ofmodified nucleoside and nucleotide sugar backbone variants known in theart include, without limitation, those having, e.g., 2′ ribosylsubstituents such as F, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3,SO2, CH3, ONO2, NO2, N3, NH2, OCH2CH2OCH3, O(CH2)2ON(CH3)2,OCH2OCH2N(CH3)2, O(C1-10 alkyl), O(C2-10 alkenyl), O(C2-10 alkynyl),S(C1-10 alkyl), S(C2-10 alkenyl), S(C2-10 alkynyl), NH(C1-10 alkyl),NH(C2-10 alkenyl), NH(C2-10 alkynyl), and O-alkyl-O-alkyl. Desirable 2′ribosyl substituents include 2′-methoxy (2′-OCH3), 2′-aminopropoxy (2′OCH2CH2CH2NH2), 2′-allyl (2′-CH2-CH═CH2), 2′-O-allyl (2′-O—CH2-CH═CH2),2′-amino (2′-NH2), and 2′-fluoro (2′-F). The 2′-substituent may be inthe arabino (up) position or ribo (down) position.

The aptamers of the invention may be made up of nucleotides and/ornucleotide analogs such as described above, or a combination of both, orare oligonucleotide analogs. The aptamers of the invention may containnucleotide analogs at positions which do not effect the function of theoligomer to bind PDGF or VEGF (or their cognate receptors).

There are several techniques that can be adapted for refinement orstrengthening of the nucleic acid Ligands binding to a particular targetmolecule or the selection of additional aptamers. One technique,generally referred to as “in vitro genetics” (see Szostak (1992) TIBS,19:89), involves isolation of aptamer antagonists by selection from apool of random sequences. The pool of nucleic acid molecules from whichthe disclosed aptamers may be isolated may include invariant sequencesflanking a variable sequence of approximately twenty to fortynucleotides. This method has been termed Selective Evolution of Ligandsby EXponential Enrichment (SELEX). Compositions and methods forgenerating aptamer antagonists of the invention by SELEX and relatedmethods are known in the art and taught in, for example, U.S. Pat. No.5,475,096 entitled “Nucleic Acid Ligands,” and U.S. Pat. No. 5,270,163,entitled “Methods for Identifying Nucleic Acid Ligands,” each of whichis specifically incorporated by reference herein in its entirety. TheSELEX process in general, and VEGF and PDGF aptamers and formulations inparticular, are further described in, e.g., U.S. Pat. Nos. 5,668,264,5,696,249, 5,670,637, 5,674,685, 5,723,594, 5,756,291, 5,811,533,5,817,785, 5,958,691, 6,011,020, 6,051,698, 6,147,204, 6,168,778,6,207,816, 6,229,002, 6,426,335, 6,582,918, the contents of each ofwhich is specifically incorporated by reference herein.

Briefly, the SELEX method involves selection from a mixture of candidateoligonucleotides and step-wise iterations of binding to a selectedtarget, partitioning and amplification, using the same general selectionscheme, to achieve virtually any desired criterion of binding affinityand selectivity. Starting from a mixture of nucleic acids, typicallycomprising a segment of randomized sequence, the SELEX method includessteps of contacting the mixture with the target under conditionsfavorable for binding, partitioning unbound nucleic acids from thosenucleic acids which have bound specifically to target molecules,dissociating the nucleic acid-target complexes, amplifying the nucleicacids dissociated from the nucleic acid-target complexes to yield aligand-enriched mixture of nucleic acids, then reiterating the steps ofbinding, partitioning, dissociating and amplifying through as manycycles as desired to yield highly specific high affinity nucleic acidligands to the target molecule.

The basic SELEX method has been modified to achieve a number of specificobjectives. For example, U.S. Pat. No. 5,707,796, entitled “Method forSelecting Nucleic Acids on the Basis of Structure,” describes the use ofthe SELEX process in conjunction with gel electrophoresis to selectnucleic acid molecules with specific structural characteristics, such asbent DNA. U.S. Pat. No. 5,763,177 entitled “Systematic Evolution ofLigands by Exponential Enrichment: Photoselection of Nucleic AcidLigands and Solution SELEX” describe a SELEX based method for selectingnucleic acid ligands containing photoreactive groups capable of bindingand/or photocrosslinking to and/or photoinactivating a target molecule.U.S. Pat. No. 5,580,737 entitled “High-Affinity Nucleic Acid LigandsThat Discriminate Between Theophylline and Caffeine,” describes a methodfor identifying highly specific nucleic acid ligands able todiscriminate between closely related molecules, which can benon-peptidic, termed Counter-SELEX. U.S. Pat. No. 5,567,588 entitled“Systematic Evolution of Ligands by EXponential Enrichment: SolutionSELEX,” describes a SELEX-based method which achieves highly efficientpartitioning between oligonucleotides having high and low affinity for atarget molecule.

The SELEX method encompasses the identification of high-affinity nucleicacid ligands containing modified nucleotides conferring improvedcharacteristics on the ligand, such as improved in vivo stability orimproved delivery characteristics. Examples of such modificationsinclude chemical substitutions at the ribose and/or phosphate and/orbase positions. SELEX process-identified nucleic acid ligands containingmodified nucleotides are described in U.S. Pat. No. 5,660,985 entitled“High Affinity Nucleic Acid Ligands Containing Modified Nucleotides,”that describes oligonucleotides containing nucleotide derivativeschemically modified at the 5- and 2′-positions of pyrimidines. U.S. Pat.No. 5,580,737, supra, describes highly specific nucleic acid ligandscontaining one or more nucleotides modified with 2′-amino (2′-NH₂),2′-fluoro (2′-F), and/or 2′-O-methyl (2′-OMe). U.S. patent applicationSer. No. 08/264,029, filed Jun. 22, 1994, entitled “Novel Method ofPreparation of Known and Novel 2′ Modified Nucleosides by IntramolecularNucleophilic Displacement,” now abandoned, describes oligonucleotidescontaining various 2′-modified pyrimidines.

The SELEX method encompasses combining selected oligonucleotides withother selected oligonucleotides and non-oligonucleotide functional unitsas described in U.S. Pat. No. 5,637,459 entitled “Systematic Evolutionof Ligands by EXponential Enrichment: Chimeric SELEX,” and U.S. Pat. No.5,683,867 entitled “Systematic Evolution of Ligands by EXponentialEnrichment: Blended SELEX,” respectively. These patents allow for thecombination of the broad array of shapes and other properties, and theefficient amplification and replication properties, of oligonucleotideswith the desirable properties of other molecules.

The SELEX method further encompasses combining selected nucleic acidligands with lipophilic compounds or non-immunogenic, high molecularweight compounds in a diagnostic or therapeutic complex as described inU.S. Pat. No. 6,011,020, entitled “Nucleic Acid Ligand Complexes,” whichis specifically incorporated by reference herein in their entirety.

The aptamer antagonists can also be refined through the use of computermodeling techniques. Examples of molecular modeling systems are theCHARMm and QUANTA programs, Polygen Corporation (Waltham, Mass.). CHARMmperforms the energy minimization and molecular dynamics functions.QUANTA performs the construction, graphic modeling and analysis ofmolecular structure. QUANTA allows interactive construction,modification, visualization, and analysis of the behavior of moleculeswith each other. These applications can be adapted to define and displaythe secondary structure of RNA and DNA molecules.

Aptamers with these various modifications can then be tested forfunction using any suitable assay for the PDGF or VEGF function ofinterest, such as a PDGF cell-based proliferation activity assay.

The modifications can be pre- or post-SELEX process modifications.Pre-SELEX process modifications yield nucleic acid ligands with bothspecificity for their SELEX target and improved in vivo stability.Post-SELEX process modifications made to 2′-OH nucleic acid ligands canresult in improved in vivo stability without adversely affecting thebinding capacity of the nucleic acid ligand.

Other modifications useful for producing aptamers of the invention areknown to one of ordinary skill in the art. Such modifications may bemade post-SELEX process (modification of previously identifiedunmodified ligands) or by incorporation into the SELEX process.

It has been observed that aptamers, or nucleic acid ligands, in general,and VEGF aptamers in particular, are most stable, and thereforeefficacious when 5′-capped and 3′-capped in a manner which decreasessusceptibility to exonucleases and increases overall stability.Accordingly, the invention is based in one embodiment, upon the cappingof aptamers in general, and anti-VEGF aptamers in particular, with a5′-5′ inverted nucleoside cap structure at the 5′ end and a 3′-3′inverted nucleoside cap structure at the 3′ end. Accordingly, theinvention provides anti-VEGF and/or anti-PDGF aptamers, i.e., nucleicacid ligands, that are capped at the 5′ end with a 5′-5-invertednucleoside cap and at the 3′ end with a 3′-3′ inverted nucleoside cap.

Certain particularly useful aptamers of the invention are anti-VEGFaptamer compositions, including, but not limited to, those having both5′-5′ and 3′-3′ inverted nucleotide cap structures at their ends. Suchanti-VEGF capped aptamers may be RNA aptamers, DNA aptamers or aptamershaving a mixed (i.e., both RNA and DNA) composition. Suitable anti-VEGFaptamer sequences of the invention include the nucleotide sequenceGAAGAAUUGG (SEQ ID NO: 15); or the nucleotide sequence UUGGACGC (SEQ IDNO: 16); or the nucleotide sequence GUGAAUGC (SEQ ID NO: 17).Particularly useful are capped anti-VEGF aptamers of the invention havethe sequence:X-5′-5′-CGGAAUCAGUGAAUGCUUAUACAUCCG-3′-3′-X  (SEQ ID NO: 18)where each C, G, A, and U represents, respectively, thenaturally-occurring nucleotides cytidine, guanidine, adenine, anduridine, or modified nucleotides corresponding thereto; X-5′-5′ is aninverted nucleotide capping the 5′ terminus of the aptamer; 3′-3′-X isan inverted nucleotide capping the 3′ terminus of the aptamer; and theremaining nucleotides or modified nucleotides are sequentially linkedvia 5′-3′ phosphodiester linkages. In some embodiments, each of thenucleotides of the capped anti-VEGF aptamer, individually carries a 2′ribosyl substitution, such as —OH (which is standard for ribonucleicacids (RNAs)), or —H (which is standard for deoxyribonucleic acids(DNAs)). In other embodiments the 2′ ribosyl position is substitutedwith an O(C₁₋₁₀ alkyl), an O(C₁₋₁₀ alkenyl), a F, an N₃, or an NH₂substituent.

In a still more particular non-limiting example, the 5′-5′ cappedanti-VEGF aptamer may have the structure:

T_(d)-5′-5′-C_(f)G_(m)G_(m)A_(r)A_(r)U_(f)C_(f)A_(m)G_(m)U_(f)G_(m)A_(m)A_(m)U_(f)G_(m)C_(f)U_(f)U_(f)A_(m)U_(f)A_(m)C_(f)A_(m)U_(f)C_(f)C_(f)G_(m)3′-3′-T_(d)(SEQ ID NO: 19)where “G_(m)” represents 2′-methoxyguanylic acid, “A_(m)” represents2′-methoxyadenylic acid, “C_(f)” represents 2′-fluorocytidylic acid,“U_(f)” represents 2′-fluorouridylic acid, “A_(r)” representsriboadenylic acid, and “T_(d)” represents deoxyribothymidylic acid.

Antisense, Ribozymes, and DNA Enzyme Antagonists

Antisense oligonucleotides and ribozymes that are targeted to PDGF andVEGF effect PDGF/VEGF inhibition by inhibiting protein translation fromthese messenger RNAs or by targeting degradation of the correspondingPDGF or VEGF mRNAs, respectively. These PDGF- and VEGF-targeted nucleicacids described above provide useful sequences for the design andsynthesis of these PDGF and VEGF ribozymes and antisenseoligonucleotides. Methods of design and synthesis of antisenseoligonucleotides and ribozymes are known in the art. Additional guidanceis provided herein.

One issue in designing specific and effective mRNA-targetedoligonucleotides (antisense ODNs) and ribozymes and antisense is that ofidentifying accessible sites of antisense pairing within the target mRNA(which is itself folded into a partially self-paired secondarystructure). A combination of computer-aided algorithms for predictingRNA pairing accessibility and molecular screening allow for the creationof specific and effective ribozymes and/or antisense oligonucleotidesdirected against most mRNA targets. Indeed several approaches have beendescribed to determine the accessibility of a target RNA molecule toantisense or ribozyme inhibitors. One approach uses an in vitroscreening assay applying as many antisense oligodeoxynucleotides aspossible (see Monia et al., (1996) Nature Med., 2:668-675; and Milner etal., (1997) Nature Biotechnol., 15:537-541). Another utilizes randomlibraries of ODNs (Ho et al., (1996) Nucleic Acids Res., 24:1901-1907;Birikh et al., (1997) RNA 3:429-437; and Lima et al., (1997) J. Biol.Chem., 272:626-638). The accessible sites can be monitored by RNase Hcleavage (see Birikh et al., supra; and Ho et al., (1998) NatureBiotechnol., 16:59-63). RNase H catalyzes the hydrolytic cleavage of thephosphodiester backbone of the RNA strand of a DNA-RNA duplex.

In another approach, involving the use of a pool of semi-random,chimeric chemically synthesized ODNs, is used to identify accessiblesites cleaved by RNase H on an in vitro synthesized RNA target. Primerextension analyses are then used to identify these sites in the targetmolecule (see Lima et al., supra). Other approaches for designingantisense targets in RNA are based upon computer assisted folding modelsfor RNA. Several reports have been published on the use of randomribozyme libraries to screen effective cleavage (see Campbell et al.,(1995) RNA 1:598-609; Lieber et al., (1995) Mol. Cell Biol., 15:540-551; and Vaish et al., (1997) Biochem., 36:6459-6501).

Other in vitro approaches, which utilize random or semi-random librariesof ODNs and RNase H may be more useful than computer simulations (Limaet al., supra). However, use of in vitro synthesized RNA does notpredict the accessibility of antisense ODNs in vivo because recentobservations suggest that annealing interactions of polynucleotides areinfluenced by RNA-binding proteins (see Tsuchihashi et al., (1993)Science, 267:99-102; Portman et al., (1994) EMBO J., 13:213-221; andBertrand and Rossi (1994) EMBO J., 13:2904-2912). U.S. Pat. No.6,562,570, the contents of which are incorporated herein by reference,provides compositions and methods for determining accessible siteswithin an mRNA in the presence of a cell extract, which mimics in vivoconditions.

Briefly, this method involves incubation of native or invitro-synthesized RNAs with defined antisense ODNs, ribozymes, orDNAzymes, or with a random or semi-random ODN, ribozyme or DNAzymelibrary, under hybridization conditions in a reaction medium whichincludes a cell extract containing endogenous RNA-binding proteins, orwhich mimics a cell extract due to presence of one or more RNA-bindingproteins. Any antisense ODN, Ribozyme, or DNAzyme, which iscomplementary to an accessible site in the target RNA will hybridize tothat site. When defined ODNs or an ODN library is used, RNase H ispresent during hybridization or is added after hybridization to cleavethe RNA where hybridization has occurred. RNase H can be present whenribozymes or DNAzymes are used, but is not required, since the ribozymesand DNAzymes cleave RNA where hybridization has occurred. In someinstances, a random or semi-random ODN library in cell extractscontaining endogenous mRNA, RNA-binding proteins and RNase H is used.

Next, various methods can be used to identify those sites on target RNAto which antisense ODNs, ribozymes or DNAzymes have bound and cleavagehas occurred. For example, terminal deoxynucleotidyltransferase-dependent polymerase chain reaction (TDPCR) may be used forthis purpose (see Komura and Riggs (1998) Nucleic Acids Res.,26:1807-11). A reverse transcription step is used to convert the RNAtemplate to DNA, followed by TDPCR. In this invention, the 3′ terminineeded for the TDPCR method is created by reverse transcribing thetarget RNA of interest with any suitable RNA dependent DNA polymerase(e.g., reverse transcriptase). This is achieved by hybridizing a firstODN primer (PI) to the RNA in a region which is downstream (i.e., in the5′ to 3′ direction on the RNA molecule) from the portion of the targetRNA molecule which is under study. The polymerase in the presence ofdNTPs copies the RNA into DNA from the 3′ end of P1 and terminatescopying at the site of cleavage created by either an antisense ODN/RNaseH, a ribozyme or a DNAzyme. The new DNA molecule (referred to as thefirst strand DNA) serves as first template for the PCR portion of theTDPCR method, which is used to identify the corresponding accessibletarget sequence present on the RNA.

For example, the TDPCR procedure may then be used, i.e., thereverse-transcribed DNA with guanosine triphosphate (rGTP) is reacted inthe presence of terminal deoxynucleotidyl transferase (TdT) to add an(rG)2-4 tail on the 3′ termini of the DNA molecules. Next is ligated adouble-stranded ODN linker having a 3′2-4 overhang on one strand thatbase-pairs with the (rG)2-4 tail. Then two PCR primers are added. Thefirst is a linker primer (LP) that is complementary to the strand of theTDPCR linker which is ligated to the (rG)2-4 tail (sometimes referred toas the lower strand). The other primer (P2) can be the same as P1, butmay be nested with respect to P1, i.e., it is complementary to thetarget RNA in a region which is at least partially upstream (i.e., inthe 3′ to 5′ direction on the RNA molecule) from the region which isbound by P1, but it is downstream of the portion of the target RNAmolecule which is under study. That is, the portion of the target RNAmolecule, which is under study to determine whether it has accessiblebinding sites is that portion which is upstream of the region that iscomplementary to P2. Then PCR is carried out in the known manner inpresence of a DNA polymerase and dNTPs to amplify DNA segments definedby primers LP and P2. The amplified product can then be captured by anyof various known methods and subsequently sequenced on an automated DNAsequencer, providing precise identification of the cleavage site. Oncethis identity has been determined, defined sequence antisense DNA orribozymes can be synthesized for use in vitro or in vivo.

Antisense intervention in the expression of specific genes can beachieved by the use of synthetic antisense oligonucleotide sequences(see, e.g., Lefebvre-d'Hellencourt et al., (1995) Eur. Cyokine Netw.,6:7; Agrawal (1996) TIBTECH, 14: 376; and Lev-Lehman et al., (1997)Antisense Therap. Cohen and Smicek, eds. (Plenum Press, New York)).Briefly, antisense oligonucleotide sequences may be short sequences ofDNA, typically 15-30 mer but may be as small as 7 mer (see Wagner etal., (1994) Nature, 372: 333) designed to complement a target mRNA ofinterest and form an RNA:AS duplex. This duplex formation can preventprocessing, splicing, transport or translation of the relevant mRNA.Moreover, certain AS nucleotide sequences can elicit cellular RNase Hactivity when hybridized with their target mRNA, resulting in mRNAdegradation (see Calabretta et al., (1996) Semin. Oncol., 23:78). Inthat case, RNase H will cleave the RNA component of the duplex and canpotentially release the AS to further hybridize with additionalmolecules of the target RNA. An additional mode of action results fromthe interaction of AS with genomic DNA to form a triple helix that maybe transcriptionally inactive.

In as a non-limiting example of, addition to, or substituted for, anantisense sequence as discussed herein above, ribozymes may be utilizedfor suppression of gene function. This is particularly necessary incases where antisense therapy is limited by stoichiometricconsiderations. Ribozymes can then be used that will target the samesequence. Ribozymes are RNA molecules that possess RNA catalytic abilitythat cleave a specific site in a target RNA. The number of RNA moleculesthat are cleaved by a ribozyme is greater than the number predicted by a1:1 stoichiometry (see Hampel and Tritz (1989) Biochem., 28: 4929-33;and Uhlenbeck (1987) Nature, 328: 596-600). Therefore, the presentinvention also allows for the use of the ribozyme sequences targeted toan accessible domain of an PDGF or VEGF mRNA species and containing theappropriate catalytic center. The ribozymes are made and delivered asknown in the art and discussed further herein. The ribozymes may be usedin combination with the antisense sequences.

Ribozymes catalyze the phosphodiester bond cleavage of RNA. Severalribozyme structural families have been identified including Group Iintrons, RNase P, the hepatitis delta virus ribozyme, hammerheadribozymes and the hairpin ribozyme originally derived from the negativestrand of the tobacco ringspot virus satellite RNA (sTRSV) (see Sullivan(1994) Investig. Dermatolog., (Suppl.) 103: 95S; and U.S. Pat. No.5,225,347). The latter two families are derived from viroids andvirusoids, in which the ribozyme is believed to separate monomers fromoligomers created during rolling circle replication (see Symons (1989)TIBS, 14: 445-50; Symons (1992) Ann. Rev. Biochem., 61: 641-71).Hammerhead and hairpin ribozyme motifs are most commonly adapted fortrans-cleavage of mRNAs for gene therapy. The ribozyme type utilized inthe present invention is selected as is known in the art. Hairpinribozymes are now in clinical trial and are a particularly useful type.In general the ribozyme is from 30-100 nucleotides in length.

Ribozyme molecules designed to catalytically cleave a target mRNAtranscript are known in the art (e.g., PDGF (SEQ ID NO: 1) or VEGF (SEQID NO:3) and can also be used to prevent translation of mRNA (see, e.g.,PCT International Pub. W090/11364; Sarver et al., (1990) Science,247:1222-1225 and U.S. Patent No. 5,093,246). While ribozymes thatcleave mRNA at site specific recognition sequences can be used todestroy particular mRNAs, the use of hammerhead ribozymes isparticularly useful. Hammerhead ribozymes cleave mRNAs at locationsdictated by flanking regions that form complementary base pairs with thetarget mRNA. The sole requirement is that the target mRNA have thefollowing sequence of two bases: 5′-UG-3′. The construction andproduction of hammerhead ribozymes is well known in the art and isdescribed more fully in Haseloff and Gerlach ((1988) Nature, 334: 585).

The ribozymes of the present invention also include RNAendoribonucleases (hereinafter “Cech-type ribozymes”) such as the onewhich occurs naturally in Tetrahymena thermophila (known as the IVS, orL-19 IVS RNA), and which has been extensively described by Thomas Cechand collaborators (see Zaug et al., (1984) Science, 224:574-578; Zaugand Cech (1986) Science, 231:470-475; Zaug, et al., (1986) Nature,324:429-433; International patent application No. W088/04300; Been andCech (1986) Cell, 47:207-216). The Cech-type ribozymes have an eightbase pair active site, which hybridizes to a target RNA sequence whereafter cleavage of the target RNA takes place. The invention encompassesthose Cech-type ribozymes, which target eight base-pair active sitesequences. While the invention is not limited to a particular theory ofoperative mechanism, the use of hammerhead ribozymes in the inventionmay have an advantage over the use of PDGF/VEGF-directed antisense, asrecent reports indicate that hammerhead ribozymes operate by blockingRNA translation and/or specific cleavage of the mRNA target.

As in the antisense approach, the ribozymes can be composed of modifiedoligonucleotides (e.g., for improved stability, targeting, etc.) and aredelivered to cells expressing the target mRNA. A useful method ofdelivery involves using a DNA construct “encoding” the ribozyme underthe control of a strong constitutive pol III or pol II promoter, so thattransfected cells will produce sufficient quantities of the ribozyme todestroy targeted messages and inhibit translation. Because ribozymes,unlike antisense molecules, are catalytic, a lower intracellularconcentration is required for efficiency.

As described above, nuclease resistance, where needed, is provided byany method known in the art that does not substantially interfere withbiological activity of the antisense oligodeoxynucleotides or ribozymesas needed for the method of use and delivery (Iyer et al., (1990) J.Org. Chem., 55: 4693-99; Eckstein (1985) Ann. Rev. Biochem., 54:367-402; Spitzer and Eckstein (1988) Nucleic Acids Res., 18: 11691-704;Woolf et al., (1990) Nucleic Acids Res., 18: 1763-69; and Shaw et al.,(1991) Nucleic Acids Res., 18: 11691-704). As described above foraptamers, non-limiting representative modifications that can be made toantisense oligonucleotides or ribozymes in order to enhance nucleaseresistance include modifying the phosphorous or oxygen heteroatom in thephosphate backbone, short chain alkyl or cycloalkyl intersugar linkagesor short chain heteroatomic or heterocyclic intersugar linkages. Theseinclude, e.g., preparing 2′-fluoridated, O-methylated, methylphosphonates, phosphorothioates, phosphorodithioates and morpholinooligomers. For example, the antisense oligonucleotide or ribozyme mayhave phosphorothioate bonds linking between four to six 3′-terminusnucleotide bases. Alternatively, phosphorothioate bonds may link all thenucleotide bases. Phosphorothioate antisense oligonucleotides do notnormally show significant toxicity at concentrations that are effectiveand exhibit sufficient pharmacodynamic half-lives in animals (seeAgarwal et al., (1996) TIBTECH, 14: 376) and are nuclease resistant.Alternatively the nuclease resistance for the AS-ODN can be provided byhaving a 9 nucleotide loop forming sequence at the 3′-terminus havingthe nucleotide sequence CGCGAAGCG. The use of avidin-biotin conjugationreaction can also be used for improved protection of AS-ODNs againstserum nuclease degradation (see Boado and Pardridge (1992) Bioconj.Chem., 3: 519-23). According to this concept the AS-ODN agents aremonobiotinylated at their 3′-end. When reacted with avidin, they formtight, nuclease-resistant complexes with 6-fold improved stability overnon-conjugated ODNs.

Other studies have shown extension in vivo of antisenseoligodeoxynucleotides (Agarwal et al., (1991) Proc. Natl. Acad. Sci.(USA) 88: 7595). This process, presumably useful as a scavengingmechanism to remove alien AS-oligonucleotides from the circulation,depends upon the existence of free 3′-termini in the attachedoligonucleotides on which the extension occurs. Therefore partialphosphorothioate, loop protection or biotin-avidin at this importantposition should be sufficient to ensure stability of theseAS-oligodeoxynucleotides.

In addition to using modified bases as described above, analogs ofnucleotides can be prepared wherein the structure of the nucleotide isfundamentally altered and that are better suited as therapeutic orexperimental reagents. An example of a nucleotide analog is a peptidenucleic acid (PNA) wherein the deoxyribose (or ribose) phosphatebackbone in DNA (or RNA) is replaced with a polyamide backbone, which issimilar to that found in peptides. PNA analogs have been shown to beresistant to degradation by enzymes and to have extended lives in vivoand in vitro. Further, PNAs have been shown to bind stronger to acomplementary DNA sequence than a DNA molecule. This observation isattributed to the lack of charge repulsion between the PNA strand andthe DNA strand. Other modifications that can be made to oligonucleotidesinclude polymer backbones, morpholino polymer backbones (see, e.g., U.S.Pat. No. 5,034,506, the contents of which are incorporated herein byreference), cyclic backbones, or acyclic backbones, sugar mimetics orany other modification including which can improve the pharmacodynamicsproperties of the oligonucleotide.

A further aspect of the invention relates to the use of DNA enzymes todecrease expression of the target mRNA as, e.g., PDGF or VEGF. DNAenzymes incorporate some of the mechanistic features of both antisenseand ribozyme technologies. DNA enzymes axe designed so that theyrecognize a particular target nucleic acid sequence, much like anantisense oligonucleotide, however much like a ribozyme they arecatalytic and specifically cleave the target nucleic acid.

There are currently two basic types of DNA enzymes, and both of thesewere identified by Santoro and Joyce (see, for example, U.S. Pat. No.6,110,462). The 10-23 DNA enzyme comprises a loop structure whichconnect two arms. The two arms provide specificity by recognizing theparticular target nucleic acid sequence while the loop structureprovides catalytic function under physiological conditions.

Briefly, to design DNA enzyme that specifically recognizes and cleaves atarget nucleic acid, one of skill in the art must first identify theunique target sequence. This can be done using the same approach asoutlined for antisense oligonucleotides. In certain instances, theunique or substantially sequence is a G/C rich of approximately 18 to 22nucleotides. High G/C content helps insure a stronger interactionbetween the DNA enzyme and the target sequence.

When synthesizing the DNA enzyme, the specific antisense recognitionsequence that targets the enzyme to the message is divided so that itcomprises the two arms of the DNA enzyme, and the DNA enzyme loop isplaced between the two specific arms.

Methods of making and administering DNA enzymes can be found, forexample, in U.S. Pat. No. 6,110,462. Similarly, methods of delivery DNAribozymes in vitro or in vivo include methods of delivery RNA ribozyme,as outlined herein. Additionally, one of skill in the art will recognizethat, like antisense oligonucleotides, DNA enzymes can be optionallymodified to improve stability and improve resistance to degradation.

RNAi Antagonists

Some embodiments of the invention make use of materials and methods foreffecting repression of VEGF and PDGF by means of RNA interference(RNAi). RNAi is a process of sequence-specific post-transcriptional generepression that can occur in eukaryotic cells. In general, this processinvolves degradation of an mRNA of a particular sequence induced bydouble-stranded RNA (dsRNA) that is homologous to that sequence. Forexample, the expression of a long dsRNA corresponding to the sequence ofa particular single-stranded mRNA (ss mRNA) will labilize that message,thereby “interfering” with expression of the corresponding gene.Accordingly, any selected gene may be repressed by introducing a dsRNAwhich corresponds to all or a substantial part of the mRNA for thatgene. It appears that when a long dsRNA is expressed, it is initiallyprocessed by a ribonuclease III into shorter dsRNA oligonucleotides ofas few as 21 to 22 base pairs in length. Accordingly, RNAi may beeffected by introduction or expression of relatively short homologousdsRNAs. Indeed the use of relatively short homologous dsRNAs may havecertain advantages as discussed below.

Mammalian cells have at least two pathways that are affected bydouble-stranded RNA (dsRNA). In the RNAi (sequence-specific) pathway,the initiating dsRNA is first broken into short interfering (si) RNAs,as described above. The siRNAs have sense and antisense strands of about21 nucleotides that form approximately 19 nucleotide si RNAs withoverhangs of two nucleotides at each 3′ end. Short interfering RNAs arethought to provide the sequence information that allows a specificmessenger RNA to be targeted for degradation. In contrast, thenonspecific pathway is triggered by dsRNA of any sequence, as long as itis at least about 30 base pairs in length. The nonspecific effects occurbecause dsRNA activates two enzymes: PKR (double-stranded RNA-activatedprotein kinase), which in its active form phosphorylates the translationinitiation factor eIF2 to shut down all protein synthesis, and 2′, 5′oligoadenylate synthetase (2′, 5′-AS), which synthesizes a molecule thatactivates RNase L, a nonspecific enzyme that targets all mRNAs. Thenonspecific pathway may represent a host response to stress or viralinfection, and, in general, the effects of the nonspecific pathway areminimized in particularly useful methods of the present invention.Significantly, longer dsRNAs appear to be required to induce thenonspecific pathway and, accordingly, dsRNAs shorter than about 30 basespairs are particular useful to effect gene repression by RNAi (see,e.g., Hunter et al., (1975) J. Biol. Chem., 250: 409-17; Manche et al.,(1992) Mol. Cell Biol., 12: 5239-48; Minks et al., (1979) J. Biol.Chem., 254: 10180-3; and Elbashir et al., (2001) Nature, 411: 494-8).

Certain double stranded oligonucleotides used to effect RNAi are lessthan 30 base pairs in length and may comprise about 25, 24, 23, 22, 21,20, 19, 18 or 17 base pairs of ribonucleic acid. Optionally, the dsRNAoligonucleotides of the invention may include 3′ overhang ends.Non-limiting exemplary 2-nucleotide 3′ overhangs may be composed ofribonucleotide residues of any type and may even be composed of2′-deoxythymidine resides, which lowers the cost of RNA synthesis andmay enhance nuclease resistance of siRNAs in the cell culture medium andwithin transfected cells (see Elbashi et al., (2001) Nature, 411:494-8).

Longer dsRNAs of 50, 75, 100 or even 500 base pairs or more may also beutilized in certain embodiments of the invention. Exemplaryconcentrations of dsRNAs for effecting RNAi are about 0.05 nM, 0.1 nM,0.5 nM, 1.0 nM, 1.5 nM, 25 nM or 100 nM, although other concentrationsmay be utilized depending upon the nature of the cells treated, the genetarget and other factors readily discernable the skilled artisan.Exemplary dsRNAs may be synthesized chemically or produced in vitro orin vivo using appropriate expression vectors. Exemplary synthetic RNAsinclude 21 nucleotide RNAs chemically synthesized using methods known inthe art (e.g., Expedite RNA phophoramidites and thymidinephosphoramidite (Proligo, Germany)). Synthetic oligonucleotides may bedeprotected and gel-purified using methods known in the art (see e.g.,Elbashir et al., (2001) Genes Dev., 15: 188-200). Longer RNAs may betranscribed from promoters, such as T7 RNA polymerase promoters, knownin the art. A single RNA target, placed in both possible orientationsdownstream of an in vitro promoter, will transcribe both strands of thetarget to create a dsRNA oligonucleotide of the desired target sequence.

The specific sequence utilized in design of the oligonucleotides maybe-any contiguous sequence of nucleotides contained within the expressedgene message of the target (e.g., of PDGF (e.g., SEQ ID NO:2) or VEGF(e.g., SEQ ID NO: 4). Programs and algorithms, known in the art, may beused to select appropriate target sequences. In addition, optimalsequences may be selected, as described additionally above, utilizingprograms designed to predict the secondary structure of a specifiedsingle stranded nucleic acid sequence and allow selection of thosesequences likely to occur in exposed single stranded regions of a foldedmRNA. Methods and compositions for designing appropriateoligonucleotides may be found in, for example, U.S. Pat. No. 6,251,588,the contents of which are incorporated herein by reference. mRNA isgenerally thought of as a linear molecule that contains the informationfor directing protein synthesis within the sequence of ribonucleotides.However, studies have revealed a number of secondary and tertiarystructures exist in most mRNAs. Secondary structure elements in RNA areformed largely by Watson-Crick type interactions between differentregions of the same RNA molecule. Important secondary structuralelements include intramolecular double stranded regions, hairpin loops,bulges in duplex RNA and internal loops. Tertiary structural elementsare formed when secondary structural elements come in contact with eachother or with single stranded regions to produce a more complexthree-dimensional structure. A number of researchers have measured thebinding energies of a large number of RNA duplex structures and havederived a set of rules which can be used to predict the secondarystructure of RNA (see e.g., Jaeger et al., (1989) Proc. Natl. Acad. Sci.(USA) 86:7706 (1989); and Turner et al., (1988) Ann. Rev. Biophys.Biophys. Chem., 17:167). The rules are useful in identification of RNAstructural elements and, in particular, for identifying single strandedRNA regions, which may represent particularly useful segments of themRNA to target for silencing RNAi, ribozyme or antisense technologies.Accordingly, particular segments of the mRNA target can be identifiedfor design of the RNAi mediating dsRNA oligonucleotides as well as fordesign of appropriate ribozyme and hammerheadribozyme compositions ofthe invention.

The dsRNA oligonucleotides may be introduced into the cell bytransfection with an heterologous target gene using carrier compositionssuch as liposomes, which are known in the art, e.g., Lipofectamine 2000(Life Technologies, Rockville Md.) as described by the manufacturer foradherent cell lines. Transfection of dsRNA oligonucleotides fortargeting endogenous genes may be carried out using Oligofectamine (LifeTechnologies). Transfection efficiency may be checked using fluorescencemicroscopy for mammalian cell lines after co-transfection of hGFPencoding pAD3 (Kehlenback et al., (1998) J. Cell. Biol., 141: 863-74).The effectiveness of the RNAi may be assessed by any of a number ofassays following introduction of the dsRNAs. These include, but are notlimited to, Western blot analysis using antibodies which recognize thetargeted gene product following sufficient time for turnover of theendogenous pool after new protein synthesis is repressed, and Northernblot analysis to determine the level of existing target mRNA.

Still further compositions, methods and applications of RNAi technologyfor use in the invention are provided in U.S. Pat. Nos. 6,278,039,5,723,750 and 5,244,805, which are incorporated herein by reference.

Receptor Tyrosine Kinase Inhibitor Antagonists

Also included in the invention are tyrosine kinase antagonists known inthe art and variants and alternatives thereto that may be obtained usingroutine skill in the art and the teachings of the art incorporatedherein by reference. The extracellular signal of PDGF (and VEGF) iscommunicated to other parts of the cell via a tyrosine kinase mediatedphosphorylation event effected by the PDGF receptor (and VEGF receptor)and which affects substrate proteins downstream of the cell membranebound signaling complex. Accordingly, antagonists acting at the receptorkinase stage of PDGF (and/or VEGF) signaling are also effective in themethod of the invention.

A number of types of tyrosine kinase inhibitors that are selective fortyrosine kinase receptor enzymes such as PDGFR or VEGFR, are known (see,e.g., Spada and Myers ((1995) Exp. Opin. Ther. Patents, 5: 805) andBridges ((1995) Exp. Opin. Ther. Patents, 5: 1245). Additionally Law andLydon have summarized the anticancer potential of tyrosine kinaseinhibitors ((1996) Emerging Drugs: The Prospect For Improved Medicines,241-260). For example, U.S. Pat. No. 6,528,526 describes substitutedquinoxaline compounds that exhibit selectively inhibit platelet-derivedgrowth factor-receptor (PDGFR) tyrosine kinase activity. The knowninhibitors of PDGFR tyrosine kinase activity includes quinoline-basedinhibitors reported by Maguire et al., ((1994) J. Med. Chem., 37: 2129),and by Dolle, et al., ((1994) J. Med. Chem., 37: 2627). A class ofphenylamino-pyrimidine-based inhibitors was recently reported byTraxler, et al., in EP 564409 and by Zimmerman et al., ((1996) Biorg.Med. Chem. Lett., 6: 1221-1226) and by Buchdunger, et al., ((1995) Proc.Nat. Acad. Sci. (USA), 92: 2558). Quinazoline derivatives that areuseful in inhibiting PDGF receptor tyrosine kinase activity includebismono- and bicyclic aryl compounds and heteroaryl compounds (see,e.g., WO 92/20642), quinoxaline derivatives (see (1994) Cancer Res., 54:6106-6114), pyrimidine derivatives (Japanese Published PatentApplication No. 87834/94) and dimethoxyquinoline derivatives (seeAbstracts of the 116th Annual Meeting of the Pharmaceutical Society ofJapan (Kanazawa), (1996), 2, p. 275, 29(C2) 15-2).

Examples of VEGFR tyrosine kinase inhibitors include cinnolinederivatives, e.g., those described in U.S. Pat. No. 6,514,971, thecontents of which are incorporated herein in their entirety. Other suchcinnoline derivatives are also known. For example, (1995) J. Med Chem.,38: 3482-7 discloses 4-(3-bromoanilino)cinnoline; (1968) J. Chem. Soc.C, (9):1152-5 discloses 6-chloro-4-phenoxycinnoline; (1984) J. KarnatakUniv., Sci., 29: 82-6 discloses certain 4-anilinocinnolines; and (1973)Indian J. Chem., 11: 211-13 discloses certain 4-phenylthiocinnolines.Furthermore, (1973) J. Karnatak Univ., 18: 25-30 discloses certain4-phenoxycinnolines, (1984) J. Kamatak Univ. Sci., 29: 82-6 disclosestwo compounds: 4-(4-methoxyanilino)-6,7-dimethoxycinnoline and4-(3-chloroanilino)-6,7-dimethoxycinnoline. Furthermore, certaincinnolines with a phenyl ring linked via a group selected from —O—, —S—,—NH— and —CH₂— at the 4-position are described in U.S. Pat. Nos.5,017,579, 4,957,925, 4,994,474, and EP 0302793 A2.

Still other related compounds for inhibition of VEGFR and/or PDGFR areavailable by screening novel compounds for their effect on the receptortyrosine kinase activity of interest using a convention assay. Effectiveinhibition by a candidate PDGFR or VEGFR small molecule organicinhibitor can be monitored using a cell-based assay system as well asother assay systems known in the art.

For example, one test for activity against VEGF-receptor tyrosine kinaseis as follows. The test is conducted using Flt-1 VEGF-receptor tyrosinekinase. The detailed procedure is as follows: 30 μl kinase solution (10ng of the kinase domain of Flt-1 (see Shibuya, et al., (1990) Oncogene,5: 519-24) in 20 mM Tris.HCl pH 7.5, 3 mM manganese dichloride (MnCl₂),3 mM magnesium chloride (MgCl₂), 10 uM sodium vanadate, 0.25 mg/mlpolyethylenglycol (PEG) 20000, 1 mM dithiothreitol and 3 ug/.mu.1poly(Glu,Tyr) 4:1 (Sigma, Buchs, Switzerland), 8 uM [³³ P]-ATP (0.2uCi), 1% dimethyl sulfoxide, and 0 to 100 μM of the compound to betested are incubated together for 10 minutes at room temperature. Thereaction is then terminated by the addition of 10 μl 0.25 Methylenediaminetetraacetate (EDTA) pH 7. Using a multichannel dispenser(LAB SYSTEMS, USA), an aliquot of 20 μl is applied to a PVDF (=polyvinyldifluoride) Immobilon P membrane (Millipore, USA), through a microtiterfilter manifold and connected to a vacuum. Following completeelimination of the liquid, the membrane is washed 4 times successivelyin a bath containing 0.5% phosphoric acid (H₃ PO₄) and once withethanol, incubated for 10 minutes each time while shaking, then mountedin a Hewlett Packard TopCount Manifold and the radioactivity measuredafter the addition of 10 μl Microscint.RTM. (beta-scintillation counterliquid). IC₅₀-values are determined by linear regression analysis of thepercentages for the inhibition of each compound in three concentrations(as a rule 0.01 μmol, 0.1 μmol, and 1 μmol). The IC₅₀-values of activetyrosine inhibitor compounds may be in the range of 0.01 μM to 100 μM.

Furthermore, inhibition of a VEGF-induced VEGFR tyrosinekinase/autophosphorylation activity can be confirmed with a furtherexperiment on cells. Briefly, transfected CHO cells, which permanentlyexpress human VEGF receptor (VEGFR/KDR), are seeded in complete culturemedium (with 10% fetal call serum (FCS) in 6-well cell-culture platesand incubated at 37° C. under 5% CO₂ until they show about 80%confluency. The compounds to be tested are then diluted in culturemedium (without FCS, with 0.1% bovine serum albumin) and added to thecells. (Controls comprise medium without test compounds). After a twohour incubation at 37° C., recombinant VEGF is added; the final VEGFconcentration is 20 ng/ml). After a further five minutes incubation at37° C, the cells are washed twice with ice-cold PBS) and immediatelylysed in 100 μl lysis buffer per well. The lysates are then centrifugedto remove the cell nuclei, and the protein concentrations of thesupernatants are determined using a commercial protein assay (BIORAD).The lysates can then either be immediately used or, if necessary, storedat −200 ° C.

A sandwich ELISA is then carried out to measure the KDR-receptorphosphorylation: a monoclonal antibody to KDR is immobilized on blackELISA plates (OptiPlate™, HTRF-96 from Packard). The plates are thenwashed and the remaining free protein-binding sites are saturated with1% BSA in PBS. The cell lysates (20 μg protein per well) are thenincubated in these plates overnight at 4° C. together with anantiphosphotyrosine antibody coupled with alkaline phosphatase (e.g.,PY20:AP from Transduction Laboratories, Lexington, Ky.). The plates arewashed again and the binding of the antiphosphotyrosine antibody to thecaptured phosphorylated receptor is then demonstrated using aluminescent AP substrate (CDP-Star, ready to use, with Emerald II;Applied-Biosystems TROPIX Bedford, Mass.). The luminescence is measuredin a Packard Top Count Microplate Scintillation Counter. The differencebetween the signal of the positive control (stimulated with VEGF orPDGF) and that of the negative control (not stimulated with VEGF orPDGF) corresponds to VEGF-induced KDR-receptor phosphorylation (=100%).The activity of the tested substances is calculated as % inhibition ofVEGF-induced KDR-receptor phosphorylation, wherein the concentration ofsubstance that induces half the maximum inhibition is defined as theED₅₀ (effective dose for 50% inhibition). Active tyrosine inhibitorcompound have ED₅₀ values in the range of 0.001 μM to 6 μM, typically0.005 μM to 0.5 μM.

Pharmaceutical Formulations and Therapeutic Administration

The anti-VEGF and anti-PDGF agents are useful in the treatment of aneovascular disorder, including psoriasis, rheumatoid arthritis, andocular neovascular disorders. Of particular interest are therapies usinga PDGF-B antagonist compound in combination with a VEGF-A antagonist tosuppress an ocular neovascular disorder such as macular degeneration ordiabetic retinopathy. Accordingly, once a patient has been diagnosed tobe at risk at developing or having a neovascular disorder, the patientis treated by administration of a PDGF antagonist in combination with aVEGF antagonist in order to block respectively the negative effects ofPDGF and VEGF, thereby suppressing the development of a neovasculardisorder and alleviating deleterious effects associated withneovascularization. The practice of the methods according to the presentinvention does not result in corneal edema. As is discussed above, awide variety of PDGF and VEGF antagonists may be used in the presentinvention.

Anti-PDGF and anti-VEGF combination therapy according to the inventionmay be performed alone or in conjunction with another therapy and may beprovided at home, the doctor's office, a clinic, a hospital's outpatientdepartment, or a hospital. Treatment generally begins at a hospital sothat the doctor can observe the therapy's effects closely and make anyadjustments that are needed. The duration of the combination therapydepends on the type of neovascular disorder being treated, the age andcondition of the patient, the stage and type of the patient's disease,and how the patient responds to the treatment. Additionally, a personhaving a greater risk of developing a neovascular disorder (e.g., adiabetic patient) may receive treatment to inhibit or delay the onset ofsymptoms. One significant advantage provided by the present invention isthat the combination of a PDGF antagonist and a VEGF antagonist for thetreatment of a neovascular disorder allows for the administration of alow dose of each antagonist and less total active antagonist, thusproviding similar efficacy with less toxicity and side effects, andreduced costs.

Administration of each antagonist of the combination therapy may be byany suitable means that results in a concentration of the antagonistthat, combined with the other antagonist, is effective for the treatmentof a neovascular disorder. Each antagonist, for example, may be admixedwith a suitable carrier substance, and is generally present in an amountof 1-95% by weight of the total weight of the composition. Thecomposition may be provided in a dosage form that is suitable forophthalmic, oral, parenteral (e.g., intravenous, intramuscular,subcutaneous), rectal, transdermal, nasal, or inhalant administration.Accordingly, the composition may be in form of, e.g., tablets, capsules,pills, powders, granulates, suspensions, emulsions, solutions, gelsincluding hydrogels, pastes, ointments, creams, plasters, deliverydevices, suppositories, enemas, injectables, implants, sprays, oraerosols. The pharmaceutical compositions containing a single antagonistor two or more antagonists may be formulated according to conventionalpharmaceutical practice (see, e.g., Remington: The Science and Practiceof Pharmacy, (20th ed.) ed. A. R. Gennaro, 2000, Lippincott Williams &Wilkins, Philadelphia, Pa. and Encyclopedia of PharmaceuticalTechnology, eds., J. Swarbrick and J. C. Boylan, 1988-2002, MarcelDekker, New York).

Combinations of PDGF and VEGF antagonists are, in one useful aspect,administered parenterally (e.g., by intramuscular, intraperitoneal,intravenous, intraocular, intravitreal, retro-bulbar, subconjunctival,subtenon or subcutaneous injection or implant) or systemically.Formulations for parenteral or systemic administration include sterileaqueous or non-aqueous solutions, suspensions, or emulsions. A varietyof aqueous carriers can be used, e.g., water, buffered water, saline,and the like. Examples of other suitable vehicles include polypropyleneglycol, polyethylene glycol, vegetable oils, gelatin, hydrogels,hydrogenated naphalenes, and injectable organic esters, such as ethyloleate. Such formulations may also contain auxiliary substances, such aspreserving, wetting, buffering, emulsifying, and/or dispersing agents.Biocompatible, biodegradable lactide polymer, lactide/glycolidecopolymer, or polyoxyethylene-polyoxypropylene copolymers may be used tocontrol the release of the active ingredients.

Alternatively, combinations of PDGF and VEGF antagonists can beadministered by oral ingestion. Compositions intended for oral use canbe prepared in solid or liquid forms, according to any method known tothe art for the manufacture of pharmaceutical compositions.

Solid dosage forms for oral administration include capsules, tablets,pills, powders, and granules. Generally, these pharmaceuticalpreparations contain active ingredients (such as a PDGF small organicmolecule antagonist and a VEGF small organic molecule antagonist)admixed with non-toxic pharmaceutically acceptable excipients. These mayinclude, for example, inert diluents, such as calcium carbonate, sodiumcarbonate, lactose, sucrose, glucose, mannitol, cellulose, starch,calcium phosphate, sodium phosphate, kaolin and the like. Bindingagents, buffering agents, and/or lubricating agents (e.g., magnesiumstearate) may also be used. Tablets and pills can additionally beprepared with enteric coatings. The compositions may optionally containsweetening, flavoring, coloring, perfuming, and preserving agents inorder to provide a more palatable preparation.

For example, the PDGF and VEGF antagonists may be administerintraocullary by intravitreal injection into the eye as well assubconjunctival and subtenon injections. Other routes of administrationinclude transcleral, retro bulbar, intraperoteneal, intramuscular, andintravenous. Alternatively, a combination of antagonists may bedelivered using a drug delivery device or an intraocular implant (seebelow).

Liquid dosage forms for oral administration include pharmaceuticallyacceptable emulsions, solutions, suspensions, syrups, and soft gelatincapsules. These forms contain inert diluents commonly used in the art,such as water or an oil medium, and can also include adjuvants, such aswetting agents, emulsifying agents, and suspending agents.

In some instances, the combination of PDGF and VEGF antagonists can alsobe administered topically, for example, by patch or by directapplication to a region, such as the epidermis or the eye, susceptibleto or affected by a neovascular disorder, or by iontophoresis.

Formulations for ophthalmic use include tablets containing the activeingredient(s) in a mixture with non-toxic pharmaceutically acceptableexcipients. These excipients may be, for example, inert diluents orfillers (e.g., sucrose and sorbitol), lubricating agents, glidants, andantiadhesives (e.g., magnesium stearate, zinc stearate, stearic acid,silicas, hydrogenated vegetable oils, or talc).

The PDGF and VEGF antagonists may be mixed together in a tablet or othervehicle, or may be partitioned. In one example, the first antagonist iscontained on the inside of the tablet, and the second antagonist is onthe outside, such that a substantial portion of the second antagonist isreleased prior to the release of the first antagonist. If desired,antagonists in a tablet form may be delivered using a drug deliverydevice (see below).

Generally, each of the antagonists should be administered in an amountsufficient to suppress or reduce or eliminate a deleterious effect or asymptom of a neovascular disorder. The amount of an active antagonistingredient that is combined with the carrier materials to produce asingle dosage will vary depending upon the subject being treated and theparticular mode of administration.

The dosage of each antagonist of the claimed combinations depends onseveral factors including the severity of the condition, whether thecondition is to be treated or prevented, and the age, weight, and healthof the person to be treated. Additionally, pharmacogenomic (the effectof genotype on the pharmacokinetic, pharmacodynamic or efficacy profileof a therapeutic) information about a particular patient may affectdosage used. Furthermore, one skilled in the art will appreciate thatthe exact individual dosages may be adjusted somewhat depending on avariety of factors, including the specific combination of PDGF and VEGFantagonists being administered, the time of administration, the route ofadministration, the nature of the formulation, the rate of excretion,the particular neovascular disorder being treated, the severity of thedisorder, and the anatomical location of the neovascular disorder (forexample, the eye versus the body cavity). Wide variations in the neededdosage are to be expected in view of the differing efficiencies of thevarious routes of administration. For instance, oral administrationgenerally would be expected to require higher dosage levels thanadministration by intravenous or intravitreal injection. Variations inthese dosage levels can be adjusted using standard empirical routinesfor optimization, which are well-known in the art. The precisetherapeutically effective dosage levels and patterns are typicallydetermined by the attending physician such as an ophthalmologist inconsideration of the above-identified factors.

Generally, when orally administered to a human, the dosage of the PDGFantagonist or VEGF antagonist is normally about 0.001 mg to about 200 mgper day, desirably about 1 mg to 100 mg per day, and more desirablyabout 5 mg to about 50 mg per day. Dosages up to about 200 mg per daymay be necessary. For administration of the PDGF antagonist or VEGFantagonist by injection, the dosage is normally about 0.1 mg to about250 mg per day, desirably about 1 mg to about 20 mg per day, or about 3mg to about 5 mg per day. Injections may be given up to about four timesdaily. Generally, when parenterally or systemically administered to ahuman, the dosage of the VEGF antagonist for use in combination with thePDGF antagonist is normally about 0.1 mg to about 1500 mg per day, orabout 0.5 mg to 10 about mg per day, or about 0.5 mg to about 5 mg perday. Dosages up to about 3000 mg per day may be necessary.

When ophthalmologically administered to a human, the dosage of the VEGFantagonist for use in combination with the PDGF antagonist is normallyabout 0.15 mg to about 3.0 mg per day, or at about 0.3 mg to about 3.0mg per day, or at about 0.1 mg to 1.0 mg per day.

For example, for ophthalmic uses, PDGF-B and VEGF-A aptamer drugsubstances are formulated in phosphate buffered saline at pH 5-7. Sodiumhydroxide or hydrochloric acid may be added for pH adjustment. In oneworking formulation, a PDGF-B aptamer and a VEGF-A aptamer, such asEYE001, are individually formulated at three different concentrations: 3mg/100 μl, 2 mg/100 μl and 1 mg/100 μl packaged in a sterile 1 ml, USPType I graduated glass syringe fitted with a sterile 27-gauge needle.The combination drug product is preservative-free and intended forsingle use by intravitreous injection only. The active ingredient isPDGF-B and VEGF-A drug substances, at 30 mg/ml, 20 mg/ml and 10 mg/mlconcentrations. The excipients are Sodium Chloride, USP; SodiumPhosphate Monobasic, Monohydrate, USP; Sodium Phosphate Dibasic,Heptahydrate, USP; Sodium Hydroxide, USP; Hydrochloric acid, USP; andWater for injection, USP. In this form the PDGF-B and VEGF-A aptamerdrug products are in a ready-to-use sterile solution provided in asingle-use glass syringe. The syringe is removed from refrigeratedstorage at least 30 minutes (but not longer than 4 hours) prior to useto allow the solution to reach room temperature. Administration of thesyringe contents involves attaching the threaded plastic plunger rod tothe rubber stopper inside the barrel of the syringe. The rubber end capis then removed to allow administration of the product. PDGF-B andVEGF-A aptamers are administered as a 100 μl intravitreal injections onthree occasions at 28 day intervals. Patients receive 3 mg/injection pervisit. The dose is reduced to 2 mg or 1 mg, and further to 0.1 mg ifnecessary.

The specific amounts of drugs administered depend on the specificcombination of components. In a desired dose combination, the ratio ofPDGF antagonist to VEGF antagonist is about 50:1 by weight, about 20:1by weight, about 10:1 by weight, or about 4:1, about 2:1, or about 1:1by weight.

A useful combination therapy includes a PDGF-B aptamer antagonist and aVEGF-A aptamer antagonist. The antagonists are used in combination in aweight ratio range from about 0.1 to about 5.0 to about 5.0 to 0.1 ofthe PDGF-B aptamer antagonist to VEGF-A aptamer antagonist. A usefulrange of these two antagonists (PDGF-B to VEGF-A antagonist) is fromabout 0.5 to about 2.0, or from about 2.0 to 0.5, while another usefulratio is from about 1.0 to about 1.0, depending ultimately on theselection of the PDGF-B aptamer antagonist and the VEGF-A aptamerantagonist.

Administration of each drug in the combination therapy can,independently, be one to four times daily for one day to one year, andmay even be for the life of the patient. Chronic, long-termadministration will be indicated in many cases. The dosage may beadministered as a single dose or divided into multiple doses. Ingeneral, the desired dosage should be administered at set intervals fora prolonged period, usually at least over several weeks, although longerperiods of administration of several months or more may be needed.

In addition to treating pre-existing neovascular disorders, thecombination therapy that includes a PDGF antagonist and VEGF antagonistcan be administered prophylactically in order to prevent or slow theonset of these disorders. In prophylactic applications, the PDGF andVEGF antagonists are administered to a patient susceptible to orotherwise at risk of a particular neovascular disorder. Again, theprecise timing of the administration and amounts that are administereddepend on various factors such as the patient's state of health, weight,etc.

In one working example, the combination of the PDGF antagonist and theVEGF antagonist is administered to a mammal in need of treatmenttherewith, typically in the form of an injectable pharmaceuticalcomposition. In the combination aspect, for example, a PDGF-B aptamerand a VEGF-A aptamer may be administered either separately or in thepharmaceutical composition comprising both. It is generally preferredthat such administration be by injection or by using a drug deliverydevice. Parenteral, systemic, or transdermal administration is alsoacceptable.

As discussed above, when the PDGF antagonist and VEGF antagonist areadministered together, such administration can be sequential in time orsimultaneous with the sequential method being one mode ofadministration. When the PDGF and VEGF antagonists are administeredsequentially, the administration of each can be by the same or differentmethods. For sequential administration, however, it is useful that themethod employ administration of the PDGF antagonist over about fiveseconds (up to about three injections) followed by sustainedadministration every six weeks for up to about nine injections per yearof a VEGF antagonist. The PDGF antagonist may be administered at thetime of each VEGF antagonist injection or may be given less often, asdetermined by the physician. Sequential administration also includes acombination where the individual antagonists may be administered atdifferent times or by different routes or both but which act incombination to provide a beneficial effect, for example, to suppress aneovascular disorder. It is also noted that administration by injectionis particularly useful.

Pharmaceutical compositions according to the invention may be formulatedto release the active PDGF and VEGF antagonists substantiallyimmediately upon administration or at any predetermined time periodafter administration, using controlled release formulations. Forexample, a pharmaceutical composition that includes at least one of eachof a PDGF antagonist and a VEGF antagonist may be provided in sustainedrelease compositions. The use of immediate or sustained releasecompositions depends on the nature of the condition being treated. Ifthe condition consists of an acute or over-acute disorder, treatmentwith an immediate release form will be typically utilized over aprolonged release composition. For certain preventative or long-termtreatments, a sustained released composition may also be appropriate.

Administration of each of the antagonists in controlled releaseformulations is useful where the antagonist, either alone or incombination, has (i) a narrow therapeutic index (e.g., the differencebetween the plasma concentration leading to harmful side effects ortoxic reactions and the plasma concentration leading to a therapeuticeffect is small; generally, the therapeutic index, TI, is defined as theratio of median lethal dose (LD₅₀) to median effective dose (ED₅₀));(ii) a narrow absorption window in the gastro-intestinal tract; or (iii)a short biological half-life, so that frequent dosing during a day isrequired in order to sustain the plasma level at a therapeutic level.

Many strategies can be pursued to obtain controlled release in which therate of release outweighs the rate of degradation or metabolism of thetherapeutic antagonist. For example, controlled release can be obtainedby the appropriate selection of formulation parameters and ingredients,including, e.g., appropriate controlled release compositions andcoatings. Examples include single or multiple unit tablet or capsulecompositions, oil solutions, suspensions, emulsions, microcapsules,microspheres, nanoparticles, patches, and liposomes. Methods forpreparing such sustained or controlled release formulations are wellknown in the art.

Pharmaceutical compositions that include a PDGF antagonist and/or a VEGFantagonist or both may also be delivered using a drug delivery devicesuch as an implant. Such implants may be biodegradable and/orbiocompatible implants, or may be non-biodegradable implants. Theimplants may be permeable or impermeable to the active agent. Ophthalmicdrug delivery devices may be inserted into a chamber of the eye, such asthe anterior or posterior chambers or may be implanted in or on thescelra, choroidal space, or an avascularized region exterior to thevitreous. In one embodiment, the implant may be positioned over anavascular region, such as on the sclera, so as to allow for transcleraldiffusion of the drug to the desired site of treatment, e.g., theintraocular space and macula of the eye. Furthermore, the site oftranscleral diffusion may be proximity to a site of neovascularizationsuch as a site proximal to the macula.

As noted above, the invention relates to combining separatepharmaceutical compositions in a pharmaceutical pack. The combination ofthe invention is therefore provided as components of a pharmaceuticalpack. At least two antagonists can be formulated together or separatelyand in individual dosage amounts. The antagonists of the invention arealso useful when formulated as salts.

The pharmaceutical pack, in general, includes (1) an amount of a PDGFantagonist, and a pharmaceutically acceptable carrier, vehicle, ordiluent in a first unit dosage form; (2) an amount of a VEGF antagonist,and a pharmaceutically acceptable carrier, vehicle, or diluent in asecond unit dosage form; and (3) a container. The container is used toseparate components and may include, for example, a divided bottle or adivided foil packet. The separate antagonist compositions may also, ifdesired, be contained within a single, undivided container. Thepharmaceutical pack may also include directions for the administrationof the separate PDGF and VEGF antagonists. The pharmaceutical pack isparticularly advantageous when the separate components are administeredin different dosage forms, are administered at different dosage levels,or when titration of the individual components of the combination isdesired by the prescribing physician. In one embodiment, thepharmaceutical pack is designed to dispense doses of the PDGF and VEGFantagonists one at a time in the order of their intended use. In anotherexample, a pharmaceutical pack is designed to contain rows of a PDGFantagonist and a VEGF antagonist placed side by side in the pack, withinstructions on the pack to convey to the user that one pair ofantagonists is to be administered. An exemplary pharmaceutical pack isthe so-called blister pack that is well known in the pharmaceuticalpackaging industry.

Effectiveness

Suppression of a neovascular disorder is evaluated by any acceptedmethod of measuring whether angiogenesis is slowed or diminished. Thisincludes direct observation and indirect evaluation such as byevaluating subjective symptoms or objective physiological indicators.Treatment efficacy, for example, may be evaluated based on theprevention or reversal of neovascularization, microangiopathy, vascularleakage or vascular edema or any combination thereof. Treatment efficacyfor evaluating suppression of an ocular neovascular disorder may also bedefined in terms of stabilizing or improving visual acuity.

In determining the effectiveness of a particular combination therapy intreating or preventing an ocular neovascular disorder, patients may alsobe clinically evaluated by an ophthalmologist several days afterinjection and at least one-month later just prior to the next injection.ETDRS visual acuities, kodachrome photography, and fluoresceinangiography are also performed monthly for the first 4 months asrequired by the ophthalmologist.

For example, in order to assess the effectiveness of combination PDGFantagonist and VEGF antagonist therapy to treat ocularneovascularization, studies are conducted involving the administrationof either single or multiple intravitreal injections of a PDGF-B aptamerin combination with a VEGF-A aptamer (for example, a PEGylated form ofEYE001) in patients suffering from subfoveal choroidalneovascularization secondary to age-related macular degenerationaccording to standard methods well known in the ophthalmologic arts. Inone working study, patients with subfoveal choroidal neovascularization(CNV) secondary to age-related macular degeneration (AMD) receive asingle intravitreal injection of a PDGF-B aptamer and a VEGF-A aptamer.Effectiveness of the combination is monitored, for example, byophthalmic evaluation. Patients showing stable or improved vision threemonths after treatment, for example, demonstrating a 3-line or greaterimprovement in vision on the ETDRS chart, are taken as receiving aneffective dosage combination of the PDGF-B aptamer and VEGF-A aptamerthat suppresses an ocular neovascular disorder.

In a working study example, patients with subfoveal CNV secondary toage-related macular degeneration and with a visual acuity worse than20/200 on the ETDRS chart receive a single intravitreous injection ofthe PDGF-B aptamer and VEGF-A aptamer. The starting dose is 0.25 mg ofeach antagonist injected once intravitreously. Dosages of 0.5 mg, 1, 2mg and 3 mg of each antagonist are also tested. Complete ophthalmicexamination with fundus photography and fluorescein angiography is alsoperformed. The combination drug product is a ready-to-use sterilesolution composed of the PDGF-B aptamer and VEGF-A aptamer dissolved in10 mM sodium phosphate and 0.9% sodium chloride buffer injection in asterile and pyrogen free 1 cc glass body syringe barrel, with a coatedstopper attached to a plastic plunger, and a rubber end cap on thepre-attached 27 gauge needle. The PDGF-B and VEGF-A aptamers aresupplied at drug concentrations of 1 mg/ml, 2.5 mg/ml, 5 mg/ml, 10mg/ml, 20 mg/ml, or 30 mg/ml for each aptamer (expressed asoligonucleotide content) to provide a 100 μl delivery volume. Atapproximately 3 months after injection of the PDGF-B and VEGF-Aaptamers, acuity studies are performed to evaluate effectiveness of thetreatment. Patients showing stable or improved vision after treatment,for example, those showing as a 3-line, or greater, increase in visionon the ETDRS chart, are taken as receiving an effective dosagecombination of PDGF-B and VEGF-A aptamers that suppresses an ocularneovascular disorder.

EXAMPLES

The following examples illustrate certain modes of making and practicingthe present invention, but are not meant to limit the scope of theinvention since alternative methods may be used to obtain similarresults.

Example 1 Corneal Neovascularization (Corneal NT)

Corneal Neovascularization is a widely used animal model that allowsclear visualization of abnormal vascular growth in the eye. The vesselsthat grow into the normally avascular cornea, can become wellestablished, making this an attractive model to study vessel regression.To induce experimental corneal NV, male C57BL/6 mice (18-20 g; CharlesRiver, Wilmington, Mass.) were anesthetized with intramuscular ketaminehydrochloride (25 mg/kg) and xylazine (10 mg/kg). NaOH (2 ul of 0.2 mM)was applied topically. The corneal and limbal epithelia were removed byapplying a rotary motion parallel to the limbus using #21 blade(Feather, Osaka, Japan). After 7 days, mice were treated withintra-peritoneal injections of 25 mg/kg of pegaptanib sodium (Macugen™(Eyetech Pharmaceuticals, New York, N.Y.), an anti-VEGF aptamer agentalso known as EYE001) twice a day or by oral administration of 50 mg/kgof Gleevec®/STI57 ((also known as CGP57148B) a2-phenylaminopyrimidine-related, tyrosine kinase-inhibiting anti-PDGFagent from Novartis Pharma AG, Basel, Switzerland) by gavage twice a dayor both for 7 days. At day 14 following corneal NV induction, micereceived 20 ug/g of fluorescein-isothiocyanate coupled concanavalin Alectin (Vector Laboratories, Burlingame, Calif.) intravenously whilstdeeply anesthetized with xylazine hydrochloride and ketaminehydrochloride. Thirty minutes later, mice eyes were enucleated, and thecorneas flat-mounted. Corneal NV was visualized using fluorescencemicroscopy and quantified using Openlab software. The percent of corneacovered by vessels was calculated as a percentage of total corneal area.

The effects of pegaptanib sodium and Gleevec on neovascularization ofthe cornea following NaOH application and injury to the epithilia of thelimbus and cornea were investigated. Animals treated with pegaptanibsodium (Macugen) showed a 19.6% (p=0.0014) decrease in vessel growth ascompared to both untreated and Gleevec treated eyes (FIG. 5). Animalstreated with pegaptanib sodium and Gleevec (Mac+Glee) exhibitedsignificantly less neovascular growth on the cornea (35.6% p<0.0001) ascompared to controls and animals treated with Gleevec alone (FIG. 5).Combination treatment was also more effective than pegaptanib sodium(Macugen) alone at reducing vessel growth (16% p<0.0145).

The results of representative corneal neovascularization experiments arealso shown in FIGS. 6 and 7. FIG. 6(D) is a photographic representationof a fluorescent-microscopic image showing effective inhibition of newblood vessel formation in combination (Mac+Gleevec)-treated corneas, ascompared to individual treatments with Macugen (FIG. 6(C)) or Gleevec(FIG. 6(B)). FIG. 6(A) is a photographic representation of afluorescent-microscopic image showing the extent of neovascularizationin a control (PEG-treated) cornea. FIG. 7 is a photographicrepresentation of a fluorescent-microscopic image showing that theindividual (FIG. 7(A) (APB5-treated) and FIG. 7(B) (Gleevec-treated))and combined treatments (FIG. 7(C)) inhibited only new vessel growth,and did not affect established blood vessels. FIG. 7(D) is aphotographic representation of a fluorescent-microscopic image showingthe extent of neovascularization in a control (PEG-treated) cornea.

Example 2 Choroidal Neovascularization (CNV)

Experimental CNV is often used as a model for Age-related Maculardegeneration (AMD). In this model, vessels of the choroid grow throughbreaks in Bruch's membrane and into the retina, similar to what isobserved in AMD patients. To induce experimental CNV, male C57BL/6 mice(18-20 g; Charles River, Wilmington, Mass.) were anesthetized withintramuscular ketamine hydrochloride (25 mg/kg) and xylazine (10 mg/kg)and the pupils were dilated with 1% tropicamide. Four burns weregenerated using diode laser photocoagulation (75-μm spot size,0.1-second duration, 90 mW, Oculight SL laser, IRIDEX, Mountain View,Calif.) and a hand-held cover slide as a contact lens. Burns localizedto the 3, 6, 9 and 12 o'clock positions of the posterior pole of theretina. Production of a bubble at the time of laser, which indicatesrupture of Bruch's membrane, is an important factor in obtainingchoroidal neovascularization, so only mice in which a bubble wasproduced for all four burns were included in the study. After 7 days,mice were treated with intraperitoneal injections of 25 mg/kg ofpegaptanib sodium twice a day or 50 mg/kg of Gleevec®/ST157 (NovartisPharma AG, Basel, Switzerland) by gavage twice a day or both for 7 days.In experiments using APB5 (an anti-mouse PDGFRb (CD140b) antibody(anti-PDGF agent) from eBioscience, San Diego, Calif.), 5 mg/kg ofantibody was administered using intra-peritoneal injections of twice aday. The area of choroidal NV lesions was measured in flat-mountedchoroid stained with PECAM. Flat-mounts were examined by fluorescencemicroscopy and quantified using Openlab software.

Eyes treated with pegaptanib sodium (Macugen™) showed a 24% (p=0.007)decrease in CNV area compared to untreated controls (FIG. 8). Incontrast, APB5-treated eyes were not significantly different to controls(6.5% decrease in CNV area compared to control). Eyes treated with bothpegaptanib sodium and APB5 showed significantly less (46% p=0.001) CNVarea as compared to control eyes or to eyes treated with eitherpegaptanib sodium (22% p=0.011) or APB5 (39.5% p<0.0001) alone (FIG. 8)

A similar trend was observed when using the PDGFRβ inhibitor. Gleevec®treated eyes showed no significant difference to control eyes (4.2%)(FIG. 9). The area of CNV in pegaptanib sodium (Macugen™) treated eyes,however, was significantly different to that of controls (27% lessp=0.0034). Importantly, animals treated with both pegaptanib sodium andGleevec (Macugen+Gleevec) exhibited the least amount of CNV (46%p<0.0001) compared to control eyes and a 19% decrease in the CNV area ascompared to pegaptanib sodium alone treated eyes (p=0.0407) (FIG. 9).

Example 3 Neonatal Mouse Model

The effect of administering pegaptanib sodium (Macugen™), and ARC-127(Archemix Corp., Cambridge, Mass.), a PEGylated, anti-PDGF aptamerhaving the sequence CAGGCUACGN CGTAGAGCAU CANTGATCCU GT (SEQ ID NO: 20)(see SEQ ID NO: 146 from U.S. Pat. No. 6,582,918, incorporated herein byreference in its entirety) having 2′-fluoro-2′-deoxyuridine at positions6, 20 and 30, 2′-fluoro-2′-deoxycytidine at positions 8, 21, 28, and 29,2′-O-Methyl-2′-deoxyguanosine at positions 9, 15, 17, and 31,2′-O-Methyl-2′-deoxyadenosine at position 22, hexaethylene-glycolphosphoramidite at “N” in positions 10 and 23, and an invertedorientation T (i.e., 3′-3′-linked) at position 32, or both on thedeveloping vessels of the retina was investigated. Neonatal C57BL/6 micewere injected daily (in the intra-peritoneal cavity) with 100 μg ofARC-127 or 100 μg of Macugen or both, starting on postnatal day 0 P0).Mice eyes were enucleated at P4. The retinal vasculature was visualizedin flatmounted retinas by immunostaining with PECAM and NG-2 or byperfusion with ConA-FITC and analyzed by fluorescence microscopy.

Injection of ARC-127 completely blocked mural cell recruitment to thedeveloping vessels of the retina. In addition, less vessel growth wasobserved at P4 as compared to the control non-treated retina. Incontrast, Macugen did not interfere with normal blood vesseldevelopment. However, mice treated with both Macugen and ARC-127exhibited similar but significantly more severe defects than micetreated with ARC-127 alone.

These results, depicted in FIG. 10, show that Macugen has no effect onthe blood vessels of the developing retina. PDGFR-B antagonist ARC-127affects vessels outgrowth and morphology. However, Macugen incombination with ARC-127 affects blood vessels more severely than eitherof them alone.

Example 4 Combination Therapy With Anti-PDGF Aptamer and Anti-VEGFAntibody

In this example, effectiveness of a combination therapy using anti-PDGFaptamers and an anti-VEGF antibody is demonstrated using the cornealneovascularization model described above. To induce experimental cornealNV, male C57BL/6 mice (18-20 g; Charles River, Wilmington, Mass.) areanesthetized with intramuscular ketamine hydrochloride (25 mg/kg) andxylazine (10 mg/kg). NaOH (2 ul of 0.2 mM) are applied topically. Thecorneal and limbal epithelia are removed by applying a rotary motionparallel to the limbus using #21 blade (Feather, Osaka, Japan). After 7days, mice are treated with intra-peritoneal injections of 25 mg/kg ofan anti-PDGF aptamer having the structure 40 KdPEG-5′-CAGGCTACGCGTAG-AGCATCATGATCCTG(iT)-3′ (SEQ ID NO: 21) (in whichiT represents that the final nucleotide is in the inverted orientation(3′-3′ linked)) in combination with 100 μg of the anti-VEGF antibody 2C3described in U.S. Pat. No. 6,342,221 (incorporated herein by reference).At day 14 following corneal NV induction, mice receive 20 μg/g offluorescein-isothiocyanate coupled concanavalin A lectin (VectorLaboratories, Burlingame, Calif.) intravenously whilst deeplyanesthetized with xylazine hydrochloride and ketamine hydrochloride.Thirty minutes later, mice eyes are enucleated, and the corneasflat-mounted. Corneal NV is visualized using fluorescence microscopy andquantified using Openlab software. The percent of cornea covered byvessels is calculated as a percentage of total corneal area. The resultsdemonstrate the efficacy of the combination therapy over individualtreatments with the anti-PDGF aptamer or anti-VEGF antibody alone.

In separate experiments, the effects of two related anti-PDGF aptamersare tested in combination with 100 μg of the anti-VEGF antibody 2C3described in U.S. Pat. No. 6,342,221. PEGylated and un-PEGylatedversions of the following two anti-PDGF aptamers are tested:

-   (i) CAGGCUACGN CGTAGAGCAU CANTGATCCU GT (SEQ ID NO: 20) (see SEQ ID    NO: 146 from U.S. Pat. No. 6,582,918, incorporated herein by    reference in its entirety) having 2′-fluoro-2′-deoxyuridine at    positions 6, 20 and 30, 2′-fluoro-2′-deoxycytidine at positions 8,    21, 28, and 29, 2′-O-Methyl-2′-deoxyguanosine at positions 9, 15,    17, and 31, 2′-O-Methyl-2′-deoxyadenosine at position 22,    hexaethylene-glycol phosphoramidite at “N” in positions 10 and 23,    and an inverted orientation T (i.e., 3′-3′-linked) at position 32;    and-   (ii) CAGGCUACGN CGTAGAGCAU CANTGATCCU GT (SEQ ID NO: 22) (see SEQ ID    NO: 87 from U.S. Pat. No. 5,723,594, incorporated herein by    reference in its entirety) having O-methyl-2-deoxycytidine at C at    position 8, 2-O-methyl-2-deoxyguanosine at Gs at positions 9, 17 and    31, 2-O-methyl-2-deoxyadenine at A at position 22,    2-O-methyl-2-deoxyuridine at position 30, 2-fluoro-2-deoxyuridine at    U at positions 6 and 20, 2-fluoro-2deoxycytidine at C at positions    21, 28 and 29, a pentaethylene glycol phosphoramidite spacer at N at    positions 10 and 23, and an inverted orientation T (i.e.,    3′-3′-linked) at position 32. Appropriate controls are provided to    detect the improved anti-neovascular effect of the combination    therapy over individual anti-PDGF aptamer or anti-VEGF antibody    treatments. The results demonstrate the efficacy of the combination    therapy over individual treatments with the anti-PDGF aptamer or    anti-VEGF antibody alone.

Example 5 Combination of Anti-PDGF Aptamer and Anti-VEGF Aptamer BlockChoroidal Neovascularization (CNV)

In this example, effectiveness of a combination therapy using anti-PDGFaptamers and anti-VEGF aptamers in blocking chorodial neovascularizationis demonstrated using the choroidal neovascularization model describedabove. Experimental CNV is often used as a model for Age-related Maculardegeneration (AMD). In this model, vessels of the choroid grow throughbreaks in Bruch's membrane and into the retina, similar to what isobserved in AMD patients. To induce experimental CNV, male C57BL/6 mice(18-20 g; Charles River, Wilmington, Mass.) are anesthetized withintramuscular ketamine hydrochloride (25 mg/kg) and xylazine (10 mg/kg)and the pupils are dilated with 1% tropicamide. Four burns are generatedusing diode laser photocoagulation (75-μm spot size, 0.1-secondduration, 90 mW, Oculight SL laser, IRIDEX, Mountain View, Calif.) and ahand-held cover slide as a contact lens. Burns localized to the 3, 6, 9and 12 o'clock positions of the posterior pole of the retina. Productionof a bubble at the time of laser, which indicates rupture of Bruch'smembrane, is an important factor in obtaining choroidalneovascularization, so only mice in which a bubble was produced for allfour burns are included in the study. After 7 days, mice are treatedwith intraperitoneal injections of 25 mg/kg of pegaptanib sodium twice aday. In experiments using anti-PDGF aptamer, 25 mg/kg of an anti-PDGFaptamer having the structure 40 KdPEG-5′-CAGGCTACGCGTAGAGCATCATGA-TCCTG(iT)-3′ (SEQ ID NO: 21) (in whichiT represents that the final nucleotide is in the inverted orientation(3′-3′ linked)) is co-administered with pegaptanib sodium. The area ofchoroidal NV lesions is measured in flat-mounted choroid stained withPECAM. Flat-mounts are examined by fluorescence microscopy andquantified using Openlab software. The results demonstrate that eyestreated with the combination therapy showed significantly less CNV areaas compared to control eyes or to eyes treated with either pegaptanibsodium or the anti-PDGF aptamer alone.

In separate experiments, the effects of two related anti-PDGF aptamersareTested in combination with the anti-VEGF treatment by intraperitonealinjections of 25 mg/kg of pegaptanib sodium twice a day. PEGylated andun-PEGylated versions of the following two anti-PDGF aptamers aretested: (i) CAGGCUACGN CGTAGAGCAU CANTGATCCU GT (SEQ ID NO: 20) (see SEQID NO: 146 from U.S. Pat. No. 6,582,918, incorporated herein byreference in its entirety) having 2′-fluoro-2′-deoxyuridine at Positions6, 20 and 30, 2′-fluoro--2′deoxycytidine at positions 8, 21, 28, and 29,2′-O-Methyl-2′-deoxyguanosine at positions 9, 15, 17, and 31,2′-O-Methyl-2′-deoxyadenosine at position 22, hexaethylene-glycolphosphoramidite at “N” in positions 10 and 23, and an invertedorientation T (i.e., 3′-3′-linked) at position 32; and

(ii) CAGGCUACGN CGTAGAGCAU CANTGATCCU GT (SEQ ID NO: 22) (see SEQ ID NO:87 from U.S. Pat. No. 5,723,594, incorporated herein by reference in itsentirety) having O-methyl-2-deoxycytidine at C at position 8,2-O-methyl-2-deoxyguanosine at Gs at positions _9, 17 and 31,2-O-methyl-2-deoxyadenine at A at position 22, 2-O-methyl-2-deoxyuridineat position 30, 2-fluoro--2-deoxyuridine at U at positions 6 and 20,2-fluoro-2-deoxycytidine at C at positions 21, 28 and 29, apentaethylene glycol phosphoramidite spacer at N at positions 10 and 23,and an inverted orientation T (i.e., 3′-3′-linked) at position 32.Appropriate controls are provided to detect the improvedanti-neovascular effect of the combination therapy over individualanti-PDGF aptamer or anti-VEGF aptamer treatments. The resultsdemonstrate the efficacy of the combination therapy in blockingchoroidal neovascularization over individual treatments with either ofthe anti-PDGF aptamers or the anti-VEGF aptamer alone.

Example 6 Corneal Neovasclarization (Corneal NV)—Regression

The corneal NV model of Example 1 was used to investigate thecombination of an anti-VEGF aptamer and anti-PDGF aptamer. After 10days, mice were treated with intra-peritoneal injections of 25 mg/kg ofpegaptanib sodium (Macugen™, Eyetech Pharmaceuticals, New York, N.Y.),an anti-VEGF aptamer agent) twice a day and/or of 50 mg/kg of ARC-127(Archemix Corp., Cambridge, Mass., an anti-PDGF aptamer having thestructure 40 Kd PEG-5′-CAGGCTACGCGTAGAGCATCATGA-TCCTG(iT)-3′ (SEQ ID NO:21) (in which iT represents that the final nucleotide is in the invertedorientation (3′-3′ linked)) once a day for 10 days. At day 20 followingcorneal NV induction, eyes were enucleated, and the corneasflat-mounted. Corneal NV was visualized using CD31 staining (BDBiosciences Pharmingen, San Diego, Calif.) and quantified usingMetamorph software. The percent of cornea covered by vessels wascalculated as a percentage of total corneal area.

The effects of pegaptanib sodium and/or ARC-127 on the regression ofneovascularization of the cornea following NaOH application and injuryto the epithilia of the limbus and cornea are depicted in FIGS. 11 and12. Animals treated with ARC-127 did not show a significant decrease invessel growth as compared to the day 20 control. The day 20 controlsshowed a 12.92% increase in corneal neovascularization when comparedwith the day 10 controls. Animals treated with pegaptanib sodium(Macugen) alone showed a 13.81% (p≦0.016) decrease in vessel growth ascompared to day 20 controls. Animals treated with pegaptanib sodium andARC-127 exhibited significantly less neovascular growth on the cornea(26.85%, p≦0.002) as compared to control.

Example 7 Corneal Neovasclarization (Corneal NV)—Regression

The corneal NV model of Example 1 was used to investigate thecombination of an anti-VEGF aptamer and an antibody against the PDGFBreceptor. After 14 days, mice were treated with intra-peritonealinjections of 25 mg/kg of pegaptanib sodium (Macugen, an anti-VEGFaptamer agent) twice a day and/or by oral administration of 50 mg/kg ofAPB5 (a polyclonal antibody against the PDGFB receptor) by gavage twicea day for 14 days. At day 28 following corneal NV induction, micereceived 20 ug/g of fluorescein-isothiocyanate coupled concanavalin Alectin (Vector Laboratories, Burlingame, Calif.) intravenously whilstdeeply anesthetized with xylazine hydrochloride and ketaminehydrochloride. Thirty minutes later, mice eyes were enucleated, and thecorneas flat-mounted. Corneal NV was visualized using fluorescencemicroscopy and quantified using Openlab software. The percent of corneacovered by vessels was calculated as a percentage of total corneal area.

The effects of pegaptanib sodium and/or APB5 on the regression ofneovascularization of the cornea following NaOH application and injuryto the epithilia of the limbus and cornea are depicted in FIG. 13.Animals treated with pegaptanib sodium (Macugen) showed an 8.3% decreasein vessel growth as compared to control. Animals treated with pegaptanibsodium and APB5 exhibited significantly less neovascular growth on thecornea (21.4%) as compared to control.

Example 8 Corneal Neovasclarization (Corneal NV)—Regression (Order ofAddition of Therapeutic Agent)

The corneal NV model of Example 1 was used to investigate the effect oforder of addition of the combination therapy using an anti-VEGF aptamerand an antibody against the PDGFB receptor. After 14 days, mice weretreated with intra-peritoneal injections of 25 mg/kg of pegaptanibsodium (Macugen, an anti-VEGF aptamer agent) twice a day and/or by oraladministration of 50 mg/kg of APB5 (eBioscience, San Diego, Calif.), apolyclonal antibody against the PDGFB receptor, by gavage twice a dayfor 7 days at different timepoints. At day 28 following corneal NVinduction, mice received 20 ug/g of fluorescein-isothiocyanate coupledconcanavalin A lectin (Vector Laboratories, Burlingame, Calif.)intravenously whilst deeply anesthetized with xylazine hydrochloride andketamine hydrochloride. Thirty minutes later, mice eyes were enucleated,and the corneas flat-mounted. Corneal NV was visualized usingfluorescence microscopy and quantified using Openlab software. Thepercent of cornea covered by vessels was calculated as a percentage oftotal corneal area and the results are depicted in FIG. 14.

The effects of pegaptanib sodium alone from day 21-28 or APB5 alone fromday 14-21 followed by no treatment showed little effect compared withcontrol on the regression of neovascularization of the cornea followingNaOH application and injury to the epithelia of the limbus and cornea.Animals treated with APB5 from day 14-21 and pegaptanib sodium from day21-28 exhibited less neovascular growth on the cornea (13.4%) ascompared to control.

Equivalents

Various modifications and variations of the described method and systemof the invention will be apparent to those skilled in the art withoutdeparting from the scope and spirit of the invention. Although theinvention has been described in connection with specific desiredembodiments, it should be understood that the invention as claimedshould not be unduly limited to such specific embodiments. Those skilledin the art will recognize or be able to ascertain using no more thanroutine experimentation, many equivalents to the specific embodiments ofthe invention described herein. Such equivalents are intended to beencompassed in the scope of the present invention.

1. A method for suppressing an ocular neovascular disorder in a patientin need thereof wherein the ocular disorder is ischemic retinopathy,iris neovascularization, intraocular neovascularization, age-relatedmacular degeneration, corneal neovascularization, retinalneovascularization, choroidal neovascularization, diabetic retinalischemia, or proliferative diabetic retinopathy, the method comprisingadministering to the patient: (i) a PDGF antagonist that is a pegylatedaptamer whose sequence is CAGGCUACGN CGTAGAGCAU CANTGATCCU GT (SEQ IDNO:23) having 2′-fluoro-2′deoxyuridine at positions 6, 20 and 30,2′-fluoro-2′deoxycytidine at positions 8, 21, 28, and 29,2′-O-Methyl-2′-deoxyguanosine at positions 9, 15, 17, and 31,2′-O-Methyl-2′-deoxyadenosine at position 22, “N” in positions 10 and 23from a hexaethylene-glycol phosphoramidite, and an inverted orientationT at position 32; and (ii) a VEGF antagonist that is pegaptanib or asalt thereof, wherein the PDGF antagonist and the VEGF antagonist areadministered simultaneously or within 90 days of each other, and whereinthe PDGF antagonist and the VEGF antagonist are administered in anamount effective to suppress the ocular neovascular disorder in thepatient.
 2. A method for treating a patient diagnosed with an ocularneovascular disorder that is ischemic retinopathy, irisneovascularization, intraocular neovascularization, age-related maculardegeneration, corneal neovascularization, retinal neovascularization,choroidal neovascularization diabetic retinal ischemia, or proliferativediabetic retinopathy, the method comprising administering to thepatient: (i) a PDGF antagonist that is a pegylated aptamer whosesequence is CAGGCUACGN CGTAGAGCAU CANTGATCCU GT (SEQ ID NO: 23) having2′-fluoro-2′deoxyuridine at positions 6, 20 and 30,2′-fluoro-2′deoxycytidine at positions 8, 21, 28, and 29,2′-O-Methyl-2′-deoxyguanosine at positions 9, 15, 17, and 31,2′-O-Methyl-2′-deoxyadenosine at position 22, “N” in positions 10 and 23from a hexaethylene-glycol phosphoramidite, and an inverted orientationT at position 32; and (ii) a VEGF antagonist that is pegaptanib or asalt thereof, wherein the PDGF antagonist and the VEGF antagonist areadministered simultaneously or within 90 days of each other, and whereinthe PDGF antagonist and the VEGF antagonist are administered in anamount effective to treat the patient.
 3. The method of claim 1 or 2,wherein said PDGF antagonist and said VEGF antagonist are administeredwithin 10 days of each other.
 4. The method of claim 3, wherein the PDGFantagonist and said VEGF antagonist are administered within 5 days ofeach other.
 5. The method of claim 4, wherein the PDGF antagonist andsaid VEGF antagonist are administered within 24 hours of each other. 6.The method of claim 5, wherein the PDGF antagonist and said VEGFantagonist are administered simultaneously.
 7. The method of claim 1,wherein the VEGF antagonist is pegaptanib sodium.
 8. The method of claim2, wherein the VEGF antagonist is pegaptanib sodium.